Myogenesis involves the complex interplay between the down-regulation of non-muscle genes and the up-regulation of muscle-specific genes. This interplay is controlled by the myogenic regulatory factors Myf5, MRF4, MyoD and myogenin. To trigger the up-regulation of these muscle-specific factors, certain environmental cues, such as the removal of serum, signal C2C12 myoblast cells to withdraw from cell cycle, fuse and activate muscle-specific genes. Here, the level of ZBP-89 (zfp148), a Krüppel-like transcription factor, has been shown to increase during myogenesis. Over-expression of ZBP-89, via adenoviral infection, led to the enhancement of the myogenic program without requiring the removal of serum. Quantitative real-time PCR and ChIP assays documented that ZBP-89 promoted the down-regulation of Pax7 coupled with the up-regulation of MRF4 and MyoD to regulate C2C12 differentiation in vitro. In addition, ZBP-89 over-expression up-regulated p21 and Rb while promoting the down-regulation of cyclinA and cyclinD1. In converse, the diminution of ZBP-89 by siRNA promoted the retention of myogenic and cell cycle regulators at myoblast levels resulting in a concomitant delay of the myogenic program. From these studies we conclude that the transcription factor ZBP-89 plays an important role in the timing of the myogenic program.