To explore the effect and the mechanism of hyperbaric oxygen therapy on testis of GK rats. Methods: Twenty-four GK rats were randomly divided into a model group, a metformin hydrochloride group and a hyperbaric oxygen group, 8 normal Wistar male rats were in the control group. The rats in the control group, the model group and the hyperbaric oxygen group received purified water 5 mL/kg once a day, while the rats in the metformin group received intragastric administration with metformin hydrochloride 250 mg/kg once a day. In addition, the rats in the hyperbaric oxygen group inhaled pure oxygen under a constant pressure (0.15 MPa) for 30 minutes. After 3-week treatment, the testis of all rats were excised for examination. For each sample, the formalin fixed testicular tissue were made into slices. Then the slices were stained by HE to observe the tissue structure. Subsequently, the apoptosis and expression level of NOS and SOD were determined by TUNEL and immunohistochemistry. Results: Under the microscopy, the testis seminiferous tubules (HE staining) of rats in the control group were full with filamentous sperm in spermatogenic cell lumen and 5- to 6-layer sperm cells. In the model group, the rats’ seminiferous tubules were atrophied, and quantity of spermatogenic cell decreased (2–3 layers). Compared with the model group, the testis structure of rats in the hyperbaric oxygen group was more integrated, and the quantity of sperm increased. The apoptosis of testis mainly occurs in the androgone. The amounts of apoptotic cells increased significantly in the model group (P<0.05), while the apoptotic cells deceased after intervention of hyperbaric oxygen (more than the control group) whose effect was similar to the metformin group. The NOS level of the rats in the model group was much higher than that in the other group, while the level of the control group was the lowest (P<0.05). The NOS expression of the hyperbaric oxygen group was weaker than the model group, but stronger than the control group (P<0.05). Moreover, the SOD expression in the hyperbaric oxygen group was stronger than the model group, but weaker than the control group. Conclusion: Increased oxygen free radicals and apoptosis is an important cause of testicular injury in diabetes. However, hyperbaric oxygen can induce the generation of SOD, remove excess oxygen free radicals, and inhibit excessive apoptosis cells so as to protect the testis tissue.