Knockout of heat shock protein Hsp72 was shown to promote chromosomal instability and increase radiation sensitivity of mouse fibroblasts. Here, we report that downregulation of Hsp72 in human tumor cells leads to suppression of a specific branch of the DNA damage response (DDR) that facilitates DNA repair following genotoxic insults, that is, reduced accumulation of the phosphorylated form of histone H2AX (gammaH2AX). This inhibition was due to decreased expression of H2AX as well as higher rate of gammaH2AX dephosphorylation. Formation of gammaH2AX and MDC1 radiation-induced foci was impaired in Hsp72-depleted cells, which in turn enhanced DNA damage, resulting in sensitization of cells to gamma-radiation and doxorubicin. These effects of Hsp72 knockdown were dependent on activation of the p53/p21-signaling pathway. Overall, permanent activation of the p53/p21 signaling in Hsp72-depleted cells specifically impaired the gammaH2AX pathway of the DDR, enhanced DNA damage following genotoxic insults, and led to further stimulation of the p53/p21 pathway, thus creating a positive feedback loop. The resulting strong induction of p21 precipitated senescence following exposure to DNA-damaging agents, thus accounting for higher sensitivity of cells to genotoxic stresses.