The cyclin D1 gene is amplified and overexpressed in human breast cancer, functioning as a collaborative oncogene. As the regulatory subunit of a holoenzyme phosphorylating Rb, cyclin D1 promotes cell cycle progression and a noncatalytic function has been described to sequester the cyclin-dependent kinase inhibitor protein p27. Cyclin D1 overexpression correlates with tumor metastasis and cyclin D1–deficient fibroblasts are defective in migration. The genetic mechanism by which cyclin D1 promotes migration and movement is poorly understood. Herein, cyclin D1 promoted cellular migration and cytokinesis of mammary epithelial cells. Cyclin D1 enhanced cellular migratory velocity. The induction of migration by cyclin D1 was abolished by mutation of K112 or deletion of NH₂-terminal residues 46 to 90. These mutations of cyclin D1 abrogated physical interaction with p27^KIP1. Cyclin D1^−/− cells were p27^KIP1 deficient and the defect in migration was rescued by p27^KIP1 reintroduction. Conversely, the cyclin D1 rescue of cyclin D1^−/− cellular migration was reversed by p27^KIP1 small interfering RNA. Cyclin D1 regulated p27^KIP1 abundance at the posttranslational level, inhibiting the Skp2 promoter, Skp2 abundance, and induced p27^KIP1 phosphorylation at Ser¹⁰. Together, these studies show cyclin D1 promotes mammary epithelial cell migration. p27^KIP1 is required for cyclin D1–mediated cellular migration. (Cancer Res 2006; 66(20): 9986-94)