Abstract Background Previous studies suggest that human RAD9 (hRad9), encoding a DNA damage checkpoint molecule, which is frequently amplified in epithelial tumor cells of breast, lung, head and neck cancer, participates in regulation of the tumor suppressor p53-dependent transactivation of pro-survival P21 ^WAF1 . This study examined the exact mechanism of the hRad9 function, especially through the phosphorylation of the C-terminus, in the transcription regulation of P21 ^WAF1 . Results The transfection of phosphorylation-defective hRAD9 mutants of C-terminus resulted in reduction of the p53-dependent P21 ^WAF1 transactivation; the knockdown of total hRad9 elicited an increased P21 ^WAF1 mRNA expression. Immunoprecipitation and a ChIP assay showed that hRad9 and p53 formed a complex and both were associated with two p53-consensus DNA-binding sequences in the 5' region of P21 ^WAF1 gene. The association was reduced in the experiment of phosphorylation-defective hRAD9 mutants. Conclusion The present study indicates the direct involvement of hRad9 in the p53-dependent P21 ^WAF1 transcriptional mechanism, presumably via the phosphorylation sites, and alterations of the hRad9 pathway might therefore contribute to the perturbation of checkpoint activation in cancer cells.