The circadian timing system (CTS) influences virtually all physiological processes, including lactation. However, the manner that the multitude of molecular clocks that make up the CTS both centrally and peripherally affect lactation performance is currently not understood. Preliminary studies from our lab led us to hypothesize clocks in the mammary play an important role in regulation of gland development. The objective of this study was to determine if decreasing abundance of CLOCK, a core component of the circadian clock mechanism, affects growth of mammary epithelial cells. For this study a mouse mammary epithelial cell line, HC11, was transfected with shRNA that targeted Clock or a negative control scramble sequence. 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). Scramble had no effect on mRNA or CLOCK protein levels. Six-day growth curve analysis revealed HC11 lines carrying shClock had 4-fold higher growth rates (P < 0.05) and reached at least 2-fold higher cell density than scramble transfected or WT HC11 cultures. To understand how CLOCK regulates growth, shClock transfected and WT HC11 cells were plated in growth medium, serum starved for 24 h, returned to growth medium and then collected every 4 h over a 48-h period. Cell cycle analysis of propidium iodide stained cells showed shClock significantly increased response to serum starvation (90.1 ± 1.1% of shClock versus 71.3 ± 3.6% of WT-HC11 in G1 phase), and affected progression through cell cycle. Two-way ANOVA showed time and cell line had significant effects (P < 0.0001) on relative expression levels of the cell cycle regulators, Ccnd1 (regulates transition from G1 to S phase), Wee1 (regulates transition from G1 to S and G2 to M phase) and Tp63 (a tumor suppressor gene). Moreover, basal mean expression of Ccnd1 was 43% greater (P = 0.002) in shClock versus WT cell lines, and Tp63 expression was depressed by ~3-fold in shClock cultures (P < 0.0001). These data support circadian clocks play a role in regulation of epithelial cell growth in the mammary gland.