The use of Doxorubicin (DOX) and its derivatives as chemotherapeutic drugs to treat cancer patients causes dilated cardiomyopathy and congestive heart failure due to DOX-induced cardiotoxicity. In this work, using wild type (HSF-1^+/+) and heat shock factor-1 knock out (HSF-1^-/-) mouse fibroblasts and embryonic rat heart-derived cardiac H9c2 cells, we show that the magnitude of protection from DOX-induced toxicity directly correlates with the level of the heat shock protein 27 (Hsp27). Western blots of normal and heat shocked cells showed the maximum expression of Hsp27 in heat shocked cardiac H9c2 cells and no Hsp27 in HSF-1^-/- cells (normal or heat shocked). Correspondingly, the cell viability, measured (MTT assay) after treating with various concentrations of DOX, was the highest in heat shocked H9c2 cells and the lowest in HSF-1^-/- cells. Depleting Hsp27 in cardiac H9c2 cells by siRNA also reduced the viability against DOX, confirming that Hsp27 does protect cardiac cells against the DOX-induced toxicity. The cells that have lower Hsp27 levels such as HSF-1^-/-, were found to be more susceptible for aconitase inactivation. Based on these results we propose a novel mechanism that Hsp27 plays an important role in protecting aconitase from DOX generated O₂^.-, by increasing SOD activity. Such a protection of aconitase by Hsp27, eliminates the catalytic recycling of aconitase released Fe(II) and its deleterious effects in cardiac cells.