Circadian rhythms in mammals are generated by a negative transcriptional feedback loop in which PERIOD (PER) is rate-limiting for feedback inhibition. Casein kinases Idelta and Iepsilon (CKIdelta/epsilon) can regulate temporal abundance/activity of PER by phosphorylation-mediated degradation and cellular localization. Despite their potentially crucial effects on PER, it has not been demonstrated in a mammalian system that these kinases play essential roles in circadian rhythm generation as does their homolog in Drosophila. To disrupt both CKIdelta/epsilon while avoiding the embryonic lethality of CKIdelta disruption in mice, we used CKIdelta-deficient Per2(Luc) mouse embryonic fibroblasts (MEFs) and overexpressed a dominant-negative mutant CKIepsilon (DN-CKIepsilon) in the mutant MEFs. CKIdelta-deficient MEFs exhibited a robust circadian rhythm, albeit with a longer period, suggesting that the cells possess a way to compensate for CKIdelta loss. When CKIepsilon activity was disrupted by the DN-CKIepsilon in the mutant MEFs, circadian bioluminescence rhythms were eliminated and rhythms in endogenous PER abundance and phosphorylation were severely compromised, demonstrating that CKIdelta/epsilon are indeed essential kinases for the clockwork. This is further supported by abolition of circadian rhythms when physical interaction between PER and CKIdelta/epsilon was disrupted by overexpressing the CKIdelta/epsilon binding domain of PER2 (CKBD-P2). Interestingly, CKBD-P2 overexpression led to dramatically low levels of endogenous PER, while PER-binding, kinase-inactive DN-CKIepsilon did not, suggesting that CKIdelta/epsilon may have a non-catalytic role in stabilizing PER. Our results show that an essential role of CKIdelta/epsilon is conserved between Drosophila and mammals, but CKIdelta/epsilon and DBT may have divergent non-catalytic functions in the clockwork as well.