Nucleosome assembly proceeds through DNA replication-coupled or replication-independent mechanisms. For skeletal myocytes, whose nuclei have permanently exited the cell cycle, replication-independent assembly is the only mode available for chromatin remodeling. For this reason, any nucleosome composition alterations accompanying transcriptional responses to physiological signals must occur through a DNA replication-independent pathway. HIRA is the histone chaperone primarily responsible for replication-independent incorporation of histone variant H3.3 across gene bodies and regulatory regions. Thus, HIRA would be expected to play an important role in epigenetically regulating myocyte gene expression. The objective of this study was to determine the consequence of eliminating HIRA from mouse skeletal myocytes. At 6 weeks of age, myofibers lacking HIRA showed no pathological abnormalities; however, genes involved in transcriptional regulation were downregulated. By 6 months of age, myofibers lacking HIRA exhibited hypertrophy, sarcolemmal perforation and oxidative damage. Genes involved in muscle growth and development were upregulated, but those associated with responses to cellular stresses were downregulated. These data suggest that elimination of HIRA produces a hypertrophic response in skeletal muscle and leaves myofibers susceptible to stress-induced degeneration.