Cyclin-dependent kinase 5 (Cdk5) is a small 33 kDa serine/threonine kinase belonging to the cdk family. It was originally identified based on the structural similarity to Cdk1 (Cdc2) and Cdk2, key regulators of eukaryotic cell cycle progression. Unlike other members of the Cdk family however, Cdk5 is not activated by cyclins, but rather by non- cyclin proteins p35 and p39. The majority of reports regarding Cdk5 relate to neuronal development, particularly in the brain, and so whether Cdk5 functions in cell division has remained a mystery. Further studies by various groups revealed additional roles of Cdk5 outside the neuronal context, including functions in cancer progression. Recently, Cdk5 was shown to play a role in modulating the cell cycle. Here in this study, we further explore the potential function of Cdk5 in cell cycle progression, particularly in non- neuronal cells. Using both siRNA and genetic approaches, we found that the absence of Cdk5 enhances cell proliferation. The loss of Cdk5 causes an upregulation of Cyclin D1 expression, while levels of the cyclin-depdent kinase inhibitor proteins p21 and p27 are reduced. Furthermore, based on flow cytometry analysis, cells lacking Cdk5 results in cells moving from G1 to S faster than normal cells, indicating a potential function in the G1/S transition. Ionizing radiation (IR) studies also reveal that the absence of Cdk5 reduces p53 phosphorylation, and maintains the presence of γH2AX foci, indicating a disruption in DNA repair process, which may cause genomic instability. Collectively, these results support the idea that Cdk5 may play an important role in regulating the cell cycle of non-neuronal cells, particularly at the G1/S transition.