There is growing evidence that the mammary circadian clock regulates milk production, but the mechanism of regulation is poorly understood. BMAL1 is a core clock gene that acts as a transcription factor when it dimerizes with CLOCK to regulate the molecular circadian rhythm. We hypothesized that BMAL1 regulates mammary cell proliferation and metabolic activity. To test this hypothesis, BMAL1 was knocked out (BMAL1-KO) of the mouse mammary epithelial cell line HC11 using CRISPR-Cas9. A monoclonal colony was selected for study based on PCR and Western blot analysis indicative of homozygous knockout of the BMAL1 gene. Growth and differentiation of BMAL1-KO was compared with the wild-type (WT) line. Two-way ANOVA found line and day had an effect on 8-d growth curve (n = 5 experiments). Log phase doubling time was not different (P > 0.05) between WT and BMAL1-KO. However, by d 6 and 8, the BMAL1-KO cells reached stationary phase at a lower cell density (P < 0.05) than WT HC11. BMAL1-KO lines exhibited lower (P < 0.05) MTT absorbance on d 2 and 4. Images of the MTT-stained cells showed denser staining within the cytoplasm of HC11 compared with BMAL1 KO. Previous studies found BMAL1 KO mice exhibited accelerated aging attributed to reactive oxygen species (ROS). Initial study with commercial assay suggests that BMAL1-KO cells have higher ROS levels, and thus build up may be causing cell death. Q-PCR analysis found BMAL1-KO cells expressed significantly lower (P < 0.05) β-casein mRNA, which was confirmed with Western blot analysis. These data support that mammary clock plays a central role in regulating metabolic activity and cellular detoxification.