Calcineurin was demonstrated to regulate the phosphorylation of threonine (T)-172 of CDK4. We further investigated how calcineurin can regulate this essential post-translational modification on CDK4. In this study, we demonstrate that calcineurin can associate predominantly with the cytoplasmic form of CDK4 in the absence of cyclin D. The inhibition of calcineurin phosphatase activity resulted in the specific increase of the phosphorylation and activity levels of CDK4 within the mitotic fraction. The association of calcineurin with CDK4 peaked during the mitotic phase of the cell cycle and coincided with reduction of CDK4 phosphorylation. Using structural mutants to CDK4, we localized the interaction site of calcineurin within the amino terminal residues of CDK4 that are important for both cyclin D and p16INK4a binding. Our data suggest that calcineurin may regulate the kinase activity of CDK4 in a cell cycle-dependent manner and may be an important component of the negative regulation of CDK4.