DNA demethylating agents are used to reverse epigenetic silencing of tumor suppressors in cancer therapeutics. Understanding of the molecular and cellular factors involved in DNA demethylation-induced gene desilencing and senescence is still limited. We have tested the involvement of two stress chaperones, Pex19p and mortalin, in 5-Aza-2′ deoxycytidine (5AZA-dC; DNA demethylating agent)-induced senescence. We found that the cells overexpressing these chaperones were highly sensitive to 5AZA-dC, and their partial silencing eliminated 5AZA-dC-induced senescence in human osteosarcoma cells. We demonstrate that these chaperones modulate the demethylation and chromatin remodeling-dependent (as accessed by p16INK4A expression) and remodeling-independent (such as activation of tumor suppressor p53 pathway) senescence response of cells. Furthermore, we found the direct interactions of 5AZA-dC with these chaperones that may alter their functions. We conclude that both mortalin and Pex19p are important mediators, prognostic indicators, and tailoring tools for 5AZA-dC-induced senescence in cancer cells.