Acrolein is a highly toxic unsaturated aldehyde which is abundant in many circumstances. People exposed to acrolein may have significant clinical relevance in human cardiotoxicity situations. Paeoniflorin (PEF) is a bioactive glucoside isolated from the roots of Paeonia lactiflora Pall. It is reported that PEF performs a beneficial role in cardiovascular system. The aim of the current research was to evaluate the potential protective effect of PEF against acrolein-induced apoptotic damage in H9c2 cardiomyocytes. This study revealed that PEF exerted a protective effect on acrolein-induced cardiomyocyte apoptosis. Furthermore, treatment with acrolein could markedly increase the levels of reactive oxygen species (ROS) and expression of cleavage of caspase-9 and caspase-3 in H9c2 cardiomyocytes, which were significantly reversed by co-treatment with PEF (100uM). These results demonstrated that PEF protects H9c2 cardiomyocytes against acrolein-induced cardiomyocyte injury via decreasing ROS production and down regulating caspases cascade reaction, indicating that PEF is a potential therapeutic agent for cardiac toxic environmental pollutant injury.