We aimed to determine the effects of ozone treatment on functional and biochemical changes in corpus cavernosum of diabetic rats. A total of 18 rats were included in the study. The rats were divided into the three groups as control, diabetes mellitus, and diabetes mellitus + ozone therapy groups. In the latter, ozone gas mixture was administered intraperitoneally for 2 weeks after the induction of experimental diabetes model. Erectile response was evaluated by determining mean intracavernosal pressure. Tissue neuronal, inducible and endothelial nitric oxide synthase levels were evaluated with commercial ELISA kits. Immunohistochemical evaluation was also performed to determine the expression levels of nitric oxide synthases semiquantatively. Mean intracavernosal pressure and intracavernosal pressure/systemic arterial blood pressure ratio were significantly higher in the diabetes mellitus + ozone therapy group than those of diabetes mellitus group (24.57 ± 6.36 mmHg vs. 5.98 ± 2.04 mmHg, p = 0.005 and 0.81 ± 0.16 vs. 0.26 ± 0.11, p = 0.0001, respectively). The level of penile tissue endothelial nitric oxide synthase was significantly higher in diabetes mellitus + ozone therapy group compared with others (19.28 ± 3.40 ng/mL vs. 13.47 ± 2.06 ng/mL and 13.28 ± 1.48 ng/mL, P = 0.01). Endothelial nitric oxide synthase expression increased significantly with ozone therapy. Our results suggest that ozone therapy may be beneficial in reducing the negative effects of diabetes on erectile dysfunction as a result of enhanced enzymatic activity in endothelial nitric oxide synthase levels.