Abstract Deregulation of translation initiation factors contributes to many pathogenic conditions, including cancer. Here, we report the definition of a novel regulatory pathway for translational initiation with possible therapeutic import in cancer. Specifically, we found that casein kinase 1ϵ (CK1ϵ) is highly expressed in breast tumors and plays a critical role in cancer cell proliferation by controlling mRNA translation. Eukaryotic translation initiation factor eIF4E, an essential component of the translation initiation complex eIF4F, is downregulated by binding the negative-acting factor 4E-BP1. We found that genetic or pharmacologic inhibition of CK1ϵ attenuated 4E-BP1 phosphorylation, thereby increasing 4E-BP1 binding to eIF4E and inhibiting mRNA translation. Mechanistic investigations showed that CK1ϵ interacted with and phosphorylated 4E-BP1 at two novel sites T41 and T50, which were essential for 4E-BP1 inactivation along with increased mRNA translation and cell proliferation. In summary, our work identified CK1ϵ as a pivotal regulator of mRNA translation and cell proliferation that acts by inhibiting 4E-BP1 function. As CK1ϵ is highly expressed in breast tumors, these findings offer an initial rationale to explore CK1ϵ blockade as a therapeutic strategy to treat cancers driven by deregulated mRNA translation. Cancer Res; 74(1); 201–11. ©2013 AACR.