A transcription suppressor element (sequence -481 to -320) containing a G-rich motif (designated GTG) and a newly identified CAT-rich motif (designated CATR) was previously shown to modulate expression of the mouse cytochrome c oxidase Vb gene during myogenesis. Here, we show that the GTG element is critical for transcription activation in both undifferentiated and differentiated myocytes. Mutations of the CATR motif abolished transcription repression in myoblasts while limiting transcription activation in differentiated myotubes, suggesting contrasting functional attributes of this DNA motif at different stages of myogenesis. Results show that the activity of the transcription suppressor motif is modulated by an orchestrated interplay between ubiquitous transcription factors: ZBP-89, YY-1, and a member of the heterogeneous nuclear ribonucleoprotein D-like protein (also known as JKTBP1) family. In undifferentiated muscle cells, GTG motif-bound ZBP-89 physically and functionally interacted with CATR motif-bound YY-1 to mediate transcription repression. In differentiated myotubes, heterogeneous nuclear ribonucleoprotein D-like protein/JKTBP1 bound to the CATR motif exclusive of YY-1 and interacted with ZBP-89 in attenuating repressor activity, leading to transcription activation. Our results show a novel mechanism of protein factor switching in transcription regulation of the cytochrome c oxidase Vb gene during myogenesis.