Abstract The BRAFV600E mutation occurs in approximately 8% of human colorectal cancers and is associated with therapeutic resistance that is due, in part, to reactivation of MEK/ERK signaling cascade. Recently, pathway analysis identified cyclin-dependent kinase 1 (CDK1) upregulation in a subset of human BRAFV600E colorectal cancers. Therefore, it was determined whether CDK1 antagonism enhances the efficacy of MEK inhibition in BRAFV600E colorectal cancer cells. BRAFV600E colorectal cancer cell lines expressing CDK1 were sensitized to apoptosis upon siRNA knockdown or small-molecule inhibition with RO-3306 (CDK1 inhibitor) or dinaciclib (CDK1, 2, 5, 9 inhibitors). Combination of RO-3306 or dinaciclib with cobimetinib (MEK inhibitor) cooperatively enhanced apoptosis and reduced clonogenic survival versus monotherapy. Cells isogenic or ectopic for BRAFV600E displayed resistance to CDK1 inhibitors, as did cells with ectopic expression of constitutively active MEK. CDK1 inhibitors induced a CASP8-dependent apoptosis shown by caspase-8 restoration in deficient NB7 cells that enhanced dinaciclib-induced CASP3 cleavage. CDK inhibitors suppressed pro-CASP8 phosphorylation at S387, as shown by drug withdrawal, which restored p-S387 and increased mitosis. In a colorectal cancer xenograft model, dinaciclib plus cobimetinib produced significantly greater tumor growth inhibition in association with a caspase-dependent apoptosis versus either drug alone. The Cancer Genome Atlas (TCGA) transcriptomic dataset revealed overexpression of CDK1 in human colorectal cancers versus normal colon. Together, these data establish CDK1 as a novel mediator of apoptosis resistance in BRAFV600E colorectal cancers whose combined targeting with a MEK/ERK inhibitor represents an effective therapeutic strategy. Implications: CDK1 is a novel mediator of apoptosis resistance in BRAFV600E colorectal cancers whose dual targeting with a MEK inhibitor may be therapeutically effective. Mol Cancer Res; 16(3); 378–89. ©2017 AACR.