Circadian clocks influence virtually all physiological processes, including lactation. Here we investigate the role of the CLOCK gene in regulation of mammary epithelial cell growth and differentiation. Comparison of mammary morphology in late pregnant wild-type and Clock19 mice, showed that gland development was negatively impacted by genetic loss of a functional timing system. To understand if these effects were due in part to loss of CLOCK function in the gland, the mouse mammary epithelial cell line, HC11, was transfected with shRNA that targeted Clock (shClock). Cells transfected with shClock expressed 70% less Clock mRNA than wild-type (WT) HC11 cultures, which resulted in significantly depressed levels of CLOCK protein (P< 0.05). HC11 lines carrying shClock had 4-fold higher growth rates (P< 0.05), and percent of cells in G1 phase was significantly higher (90.1± 1.1% of shClock versus 71.3 ± 3.6% of WT-HC11) following serum starvation. Q-PCR analysis showed shClock had significant effects (P< 0.0001) on relative expression levels of Ccnd1, Wee1 and Tp63. Q-PCR analysis of effect of shClock on Fasn and Cdh1 expression in undifferentiated cultures, and cultures treated 96 h with dexamethasone, insulin and prolactin (differentiated), found levels were reduced by 2-fold and 3-fold, respectively (P< 0.05), in shClock line relative to WT cultures. Abundance of CDH1 and TP63 proteins were significantly reduced in cultures transfected with shClock. These data support CLOCK plays a role in regulation of epithelial cell growth and differentiation in the mammary gland.