Circadian disruption has multiple pathological consequences, but the underlying mechanisms are largely unknown. To address such mechanisms, we subjected transformed cultured cells to chronic circadian desynchrony (CCD), mimicking a chronic jet-lag scheme, and assayed a range of cellular functions. The results indicated a specific circadian clock–dependent increase in cell proliferation. Transcriptome analysis revealed up-regulation of G1/S phase transition genes (myelocytomatosis oncogene cellular homolog [Myc], cyclin D1/3, chromatin licensing and DNA replication factor 1 [Cdt1]), concomitant with increased phosphorylation of the retinoblastoma (RB) protein by cyclin-dependent kinase (CDK) 4/6 and increased G1-S progression. Phospho-RB (Ser807/811) was found to oscillate in a circadian fashion and exhibit phase-shifted rhythms in circadian desynchronized cells. Consistent with circadian regulation, a CDK4/6 inhibitor approved for cancer treatment reduced growth of cultured cells and mouse tumors in a time-of-day–specific manner. Our study identifies a mechanism that underlies effects of circadian disruption on tumor growth and underscores the use of treatment timed to endogenous circadian rhythms.