This study was designed to investigate the effect of pterostilbene (PTS) on cardiac oxidative stress in vitro, as this is a simple and promising methodology to study cardiac disease. Cardiac myoblasts (H9c2 cells) and homogenised cardiac tissue were incubated with the PTS and cyclodextrin (PTS + HPβCD) complex for 1 and 24 h, respectively, at concentrations of 50μM for the cells and 25 and 50μM for cardiac tissue. The PTS + HPβCD complex was used to increase the solubility of PTS in water. After the pretreatment period, cardiomyoblasts were challenged with hydrogen peroxide (6.67μM) for 10 min, while cardiac tissue was submitted to a hydroxyl radical generator system (30 min). Cellular viability, oxidative stress biomarkers (e.g. total reactive oxygen species (ROS), carbonyl assay and lipoperoxidation) and the antioxidant response (e.g. sulfhydryl and the antioxidant enzyme activities of superoxide dismutase, catalase and glutathione peroxidase) were evaluated. In cardiomyoblasts, the PTS + HPβCD complex (50μM) increased cellular viability. Moreover, the PTS + HPβCD complex also significantly increased sulfhydryl levels in the cells submitted to an oxidative challenge. In cardiac tissue, lipid peroxidation, carbonyls and ROS levels were significantly increased in the groups submitted to oxidative damage, while the PTS + HPβCD complex significantly reduced ROS levels in these groups. In addition, the PTS + HPβCD complex also provoked increased catalase activity in both experimental protocols. These data suggest that the PTS + HPβCD complex may play a cardioprotective role through a reduction of ROS levels associated with an improved antioxidant response.