Circadian clocks synchronize internal physiology to the environment, thus understanding role in regulation of lactation may lead to development of noninvasive approaches to improve production efficiency of dairy animals. Circadian clocks are present in all mammalian cells. The BMAL1 and CLOCK genes are core components of clocks, functioning together as a transcription factor. In the mammary gland, abundance of BMAL1-CLOCK increases during the transition from pregnancy to lactation and upon lactogen-induced differentiation of mammary cells in culture. We hypothesize that in the mammary BMAL1-CLOCK regulates differentiation and milk synthesis. Our objective was to elucidate the effect of decreasing CLOCK abundance on expression of markers of differentiation (e-cadherin- CDH1) and metabolic output (fatty acid synthase- FASN; β-casein- CSN2) in a mouse mammary epithelial cell line, HC11. To decrease CLOCK abundance, HC11 cells were transfected with shRNA specific for Clock or a scramble sequence (negative control), and clonal lines were established. Cells transfected with shClock had a 70% reduction in Clock mRNA, which resulted in significantly reduced (P < 0.05) CLOCK protein abundance relative to wild-type cultures. Abundance of CLOCK in scramble-line was not different from wild-type. RNA and protein were collected from undifferentiated cultures, and cultures treated 96 h with dexamethasone, insulin and prolactin (differentiated). Gene expression was analyzed using RT-qPCR. Two-way ANOVA showed cell line (wild-type, scramble, shClock) and state of differentiation had a significant effect (P < 0.05) on relative expression of Fasn, Csn2 and Cdh1. Post-hoc analysis revealed Fasn was significantly reduced by 2-fold (P < 0.05) in shClock treatments relative to wild-type cultures. Cdh1 was also reduced more than 3-fold in shClock lines relative to wild-type cultures (P < 0.05). Moreover, Western blot analysis showed abundance of CDH1 protein was significantly reduced in cultures transfected with shClock. shClock sequence did not have a significant effect on Csn2 expression. In conclusion CLOCK regulates differentiation markers and Fasn, future studies are needed to understand how factors affect mammary clocks and the relationship to dairy performance.