Rationale: Radioresistance is considered the main cause of local relapse in lung cancer. However, the molecular mechanisms of radioresistance remain poorly understood. This study investigates the role of CDK16 in radioresistance of human lung cancer cells. Methods: The expression levels of CDK16 were determined by immunohistochemistry in lung cancer tissues and adjacent normal lung tissues. Immunoprecipitation assay and GST pulldown were utilized to detect the protein-protein interaction. The phosphorylation of p53 was evaluated by in vitro kinase assay. Poly-ubiquitination of p53 was examined by in vivo ubiquitination assay. Cell growth and apoptosis, ROS levels and DNA damage response were measured for functional analyses. Results: We showed that CDK16 is frequently overexpressed in lung cancer cells and tissues, and high levels of CDK16 are correlated with lymph node stage and poor prognosis in lung cancer patients. Furthermore, we provided evidence that CDK16 binds to and phosphorylates p53 at Ser315 site to inhibit transcriptional activity of p53. Moreover, we uncovered that this phosphorylation modification accelerates p53 degradation via the ubiquitin/proteasome pathway. Importantly, we demonstrated that CDK16 promotes radioresistance by suppressing apoptosis and ROS production as well as inhibiting DNA damage response in lung cancer cells in a p53-dependent manner. Conclusion: Our findings suggest that CDK16 negatively modulates p53 signaling pathway to promote radioresistance, and therefore represents a promising therapeutic target for lung cancer radiotherapy.