The small heat shock protein Hsp27 is expressed at high levels in many tumors and provides protection against anticancer drugs. Here, we show that expression of recombinant Hsp27 at elevated levels leads to protection of MCF10A human mammary epithelial cells from doxorubicin. The protection was associated with suppression of the doxorubicin-induced senescence, where Hsp27 inhibited p53-mediated induction of p21, the major regulator of the senescence program. Similarly, Hsp27 inhibited accumulation of p21 and suppressed senescence in response to the p53 activator nutlin-3, indicating that Hsp27 has a general effect on the p53 pathway. In line with these findings, down-regulation of Hsp27 in HCT116 human colon carcinoma cells that express this heat shock protein at high levels caused senescence in a population of cells and sensitized the rest of the cells to doxorubicin-induced senescence (at low doses) or apoptosis (at high doses of doxorubicin). Induction of senescence by Hsp27 down-regulation associated with activation of the p53 pathway and induction of p21. Interestingly, depletion of Hsp27 caused neither significant proteotoxic nor genotoxic stress, and therefore this heat shock protein seems to have a specific effect on the p53 signaling. Indeed, Hsp27 down-regulation was associated with destabilization of HDM2 and stabilization of p53. These data suggest that Hsp27 may play a general role in regulation of cellular senescence by modulating the p53 pathway.