Cyclin-dependent kinase (cdk)-3, a member of the cdk family of kinases, plays a critical role in cell cycle regulation and is involved in G(0)-G(1) and G(1)-S cell cycle transitions. However, the role of cdk3 in cell proliferation, as well as cell transformation, is not yet clearly understood. Here, we report that the protein expression level of cdk3 is higher in human cancer cell lines and human glioblastoma tissue compared with normal brain tissue. Furthermore, we found that cdk3 phosphorylates activating transcription factor 1 (ATF1) at serine 63 and enhances the transactivation and transcriptional activities of ATF1. Results also indicated that siRNA directed against cdk3 (si-cdk3) suppresses ATF1 activity, resulting in inhibition of proliferation and growth of human glioblastoma T98G cells in soft agar. Importantly, we showed that cdk3 enhances epidermal growth factor-induced transformation of JB6 Cl41 cells and si-cdk3 suppresses Ras(G12V)/cdk3/ATF1-induced foci formation in NIH3T3 cells. These results clearly showed that the cdk3-ATF1 signaling axis is critical for cell proliferation and transformation.