A mammalian basic helix-loop-helix protein known variably as Stra13, Sharp2, and Dec1 has been implicated in cell activation, proliferation, and differentiation. Indeed, Stra13 null mice develop age-induced autoimmunity as a result of impaired T-lymphocyte activation, leading ultimately to the accumulation of autoreactive T-cells and B-cells. Stra13 is expressed in embryonic as well as adult tissues derived from neuroectoderm, mesoderm, and endoderm and has been associated with response to hypoxia, suggesting a complex role for this protein and the highly related Sharp1/Dec2 protein in homeostatic regulation. Whereas Stra13 is known to regulate many important cellular functions and is known to cross-regulate biological responses to other basic helix-loop-helix containing transcription factors, including c-Myc and USF, it is unclear if this protein binds directly to DNA. Indeed, the basic domain of Stra13 contains a proline residue at an unprecedented position. Herein, we have determined that Stra13 binds with high affinity to CACGTG class B E-box elements as a homodimer with preference for elements preceded by T and/or followed by A residues. In addition, transient transfection experiments reveal that Stra13 represses transcription when bound to these and related sites. Our data suggest that Stra13 regulates cellular functions through antagonism of E-box activator proteins and also through active repression from E-box elements.