Myoblast fusion is critical for proper muscle growth and regeneration. During myoblast fusion, the localization of some molecules is spatially restricted; however, the exact reason for such localization is unknown. Creatine kinase B (CKB), which replenishes local ATP pools, localizes near the ends of cultured primary mouse myotubes. To gain insights into the function of CKB, we performed a yeast two-hybrid screen to identify CKB-interacting proteins. We identified molecules with a broad diversity of roles, including actin polymerization, intracellular protein trafficking, and alternative splicing, as well as sarcomeric components. In-depth studies of α-skeletal actin and α-cardiac actin, two predominant muscle actin isoforms, demonstrated their biochemical interaction and partial colocalization with CKB near the ends of myotubes in vitro. In contrast to other cell types, specific knockdown of CKB did not grossly affect actin polymerization in myotubes, suggesting other muscle-specific roles for CKB. Interestingly, knockdown of CKB resulted in significantly increased myoblast fusion and myotube size in vitro, whereas knockdown of creatine kinase M had no effect on these myogenic parameters. Our results suggest that localized CKB plays a key role in myotube formation by limiting myoblast fusion during myogenesis. formation, growth, and repair of multinucleated skeletal muscle cells are dependent on fusion of progenitor myoblasts (1). In spite of the importance of myoblast fusion, the molecular mechanisms underlying this process are incompletely understood. Cell culture models have been valuable for defining the various stages of myogenesis. During myogenesis, myoblasts differentiate into elongated myocytes, which migrate and adhere to one another. At cell-cell contact sites, a high density of adhesion molecules is found (77). Following adhesion, downstream signaling pathways are activated, leading to localized actin changes at cell-cell contact sites (52). Next, membrane union takes place, leading to fusion of several differentiated myoblasts with one another both in an end-to-end and a perpendicular orientation to each other (47), to form nascent myotubes with few nuclei. Finally, differentiated myoblasts most commonly fuse with the ends of nascent myotubes (52) to generate mature myotubes containing many nuclei. A variety of extracellular, cell surface, and intracellular molecules act to finely coordinate the cellular and molecular events that influence the ability of mammalian myoblasts to fuse (1, 65). During myotube formation, some cell surface (ADAM12, integrins, kindlin-2, cadherins), membrane (cholesterol, phosphatidylserine), and intracellular (actin, β-catenin, diacyglycerol kinase, EB3, nonmuscle myosin 2A, myoferlin, Rac1, syntrophin) molecules become spatially restricted to specific cellular domains (2, 8, 13, 16–18, 37, 44, 47, 62, 73, 76, 77). A number of these molecules are localized to cell-cell contact sites in opposing muscle cells, whereas other molecules localize to cell-cell contact sites between two muscle cells but in only one of the cells (50). Although the exact reasons for such localization during myogenesis are unknown, understanding why some molecules localize in this manner may provide valuable insights into the process of myoblast fusion. We previously reported that creatine kinase B (CKB), the brain isoform of cytosolic CK enzymes, is prominently localized to myotube ends in vitro (49). CKB catalyzes the transfer of a phosphate group from phosphocreatine to ADP, thereby replenishing local cellular ATP at sites of high ATP turnover (81). These findings suggested that the ends of myotubes are sites of high ATP demand. However, the molecules that require ATP generated by CKB for their cellular function are unknown. Such molecules may be functionally important for myogenesis. Interestingly, CKB can also localize to specific cellular regions in nonmuscle cells. CKB transiently accumulates in membrane ruffles of astrocytes and fibroblasts during cell spreading and migration, and ablation of CKB activity negatively affects these two processes (36). In addition, CKB is transiently recruited to the phagocytic cup of macrophages during phagocytosis, where inhibition of CKB activity diminishes actin accumulation (35). Finally, CKB localizes to inner ear hair cells, and CKB-deficient mice exhibit hearing loss (63). Together, these studies indicate CKB activity is required at specific cellular locations in some cell types for various functions. Further examination of CKB function and localization specifically in muscle cells could enhance our understanding of the mechanisms underlying myotube formation. To gain insights into the function of CKB at myotube ends, we first identified CKB-interacting proteins using a yeast two-hybrid screen. Subsequently, in-depth studies of two of these CKB-interacting proteins, α-skeletal actin and α-cardiac actin, predominant muscle actin isoforms, demonstrated their biochemical interaction and partial colocalization with CKB near the ends of mouse myotubes in vitro. Finally, we used small-interfering RNA (siRNA) to decrease the levels of CKB and observed a significant increase in myoblast fusion and myotube size in cultured primary mouse muscle cells. Our results suggest CKB plays a key role in myotube formation by limiting myoblast fusion during myogenesis.