In contrast to conventional cyclin-dependent kinases that are important for mitotic cell division, cyclin-dependent kinase 5 (CDK5) is predominantly activated in post-mitotic cells and is involved in various cellular events. The kinase activity of CDK5 is tightly regulated by specific activators including p35, p39, and cyclin I (CCNI). Here we show that cyclin I-like (CCNI2), a homolog of CCNI, interacts with CDK5 and activates the kinase activity of CDK5. Different from CCNI, which colocalizes with CDK5 in the nuclei in transfected cells, CCNI2 mainly retains CDK5 in the cytoplasm as well as on the cell membrane. Furthermore, although the expression level of CCNI2 mRNA and CCNI2 protein do not change significantly during cell cycle, depletion of CCNI2 with siRNA affects cell cycle progression as well as cell proliferation. In conclusion, our data strongly suggest that CCNI2 is a novel CDK5 activator and is involved in cell cycle regulation.