Introduction: Vasostatin-1 (VS-1) has been suggested in protecting hypoxia/reoxygenation (H/R) injuries in isolated hearts. However, the molecular mechanisms remained to be elucidated. Methods: Cardiomyocytes were treated with recombinant Ad-VS-1 adenoviral vector before H/R. Cell viability was studied using MTT methods and annexin V-FITC flow cytometry. Intracellular oxidative stress was measured by superoxide dismutase (SOD) and malondialdehyde (MDA), and inflammatory reactions by enzyme-linked immunosorbent assay (ELISA). Measurement of myocardial nitrous oxide synthase (NOS) was determined by serum nitric oxide (NO) concentrations using nitrite reductase and endothelial nitric oxide synthase (eNOS) by Western blotting. Inhibitors of the NOS system, including hemoglobin and KT5823, were applied to verify the results. Results: In comparison of the blank group, cardiac myocytes overexpressing VS-1 showed significant decrease in apoptosis, intracellular oxidative stress, and inflammatory reactions (P < 0.05). In addition, serum NO concentrations and expression of eNOS were notably enhanced (P < 0.05). These protective effects of VS-1 were suppressed in the presence of apoptosis-inducing agents. Conclusions: Overexpression of VS-1 in cardiomyocytes could limit the H/R injuries at molecular levels. The protective effects were independent of endothelial cell function, suggestive of a potential therapeutic target for patients with myocardial ischemia in the future.