3β-Hydroxy-5,6-secocholestan-6-al (cholesterol secoaldehyde or ChSeco), an oxysterol known to be formed in ozone- and singlet oxygen-mediated oxidations of cholesterol, has been detected in the atherosclerotic plaque and in the brain of patients suffering from Alzheimer’s disease and Lewy body dementia. Previously, we have shown that, in H9c2 cardiomyoblasts, ChSeco induces oxidative stress followed by apoptosis involving both intrinsic and extrinsic signaling pathways. In the present study, we investigated the nature of reactive oxygen species (ROS) and its associated redox signaling in H9c2 cells upon treatment with ChSeco. Both catalase and deferoxamine, which lowered intracellular ROS, were found to alleviate the ChSeco-induced cytotoxicity. ChSeco-treated H9c2 cells showed a significant decrease in the intracellular catalase activity, suggesting the involvement of H2O2 in the associated cytotoxicity. Additionally, in ChSeco-exposed cells, there was a marked increase in lipid peroxidation and pre-treatment with SB 203580 (p38 MAPK inhibitor) and MEK1/2 inhibitor (ERK1/2 and JNK inhibitor) rendered protection against the cytotoxicity. An early increase in the expression of p-SAPK/JNK or delayed p38 MAPK did not alter ATF-2 but decreased c-Jun expression in these cells. Overall, these findings are consistent with MAPK signaling resulting from increased cellular H2O2 in ChSeco-induced cytotoxicity in cardiomyoblasts.