The p53 tumor suppressor plays a key role in induction and maintenance of cellular senescence but p53-regulated response to stress in senescent cells is poorly understood. Here, we use the small-molecule MDM2 antagonist, nutlin-3a, to selectively activate p53 and probe functionality of the p53 pathway in senescent human fibroblasts, WI-38. Our experiments revealed overall reduction in nutlin-induced transcriptional activity of nine p53 target genes and four p53-regulated microRNAs, indicating that not only p53 protein levels but also its ability to activate transcription are altered during senescence. Addition of nutlin restored doxorubicin-induced p53 protein and transcriptional activity in senescent cells to the levels in early passage cells but only partially restored its apoptotic activity, suggesting that changes in both upstream and downstream p53 signaling during senescence are responsible for attenuated response to genotoxic stress.