Increased CLP36 expression has been found to be closely associated with breast cancer progression. However, whether and how it contributes to malignant behavior of breast cancer cells were not known. We show here that CLP36 is critical for promoting breast cancer cell migration and invasion in vitro and metastasis in vivo, whereas it is dispensable for breast cell proliferation and anchorage-independent growth in vitro and tumor growth in vivo. CLP36 interacted with both α-actinin-1 and -4 in breast cancer cells. Depletion of either α-actinin-1 or -4 inhibited breast cancer cell migration. Furthermore, mutations inhibiting the α-actinin-binding activity abolished the ability of CLP36 to promote breast cancer cell migration. Finally, depletion of CLP36 or disruption of the CLP36-α-actinin complex in breast cancer cells substantially inhibited Cdc42 activation, cell polarization and migration. Our results identify CLP36 as an important regulator of breast cancer cell migration and metastasis, and shed light on how increased CLP36 expression contributes to the progression of breast cancer.