Testicular dysfunction and infertility are serious complications of diabetes mellitus (DM). L-Arginine (L-Arg) is a semi essential amino acid with various biological and metabolic functions. The molecular mechanisms of L-Arg on testicular dysfunction caused by DM remain elusive. This study aimed to assess the potential protective effect of L-Arg in diabetic testis and its possible mechanisms. 24 adult male Wistar albino rats were randomly divided into four groups: CON, L-Arg that received 1 g/kg body weight of L-Arg orally for 4 weeks, DM that fed a high fat diet followed by an injection of 30 mg/kg streptozotocin intraperitoneally, and L-Arg-treated DM that were diabetic and administered L-Arg. DM decreased relative testicular weight, reduced serum testosterone, and impaired semen parameters. Reduced total antioxidant capacity (TAC), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px), in addition to increased transforming growth factor B1 (TGF-β1) and nitric oxide (NO) levels, were found in the testicular tissue. This was associated with severe degenerative changes in the seminiferous tubules and interstitial cells of Leydig, reduction of Johnsen's score, significantly increased expression of both inducible nitric oxide synthase (iNOS) and caspase-3, and reduced zonula occludens (ZO)− 1 expression. Ultrastructurally, disrupted intercellular junctions and degeneration of interstitial cells of Leydig were observed. In contrast, treatment of diabetic animals with L-Arg increased TAC, SOD and GSH-Px, decreased TGF-β1 and NO levels, downregulated iNOS and caspase-3 expression, upregulated ZO-1 expression, and maintained the integrity of the Sertoli cell junctions. Hence, L-Arg restored the normal testicular structure and function via its antioxidant, antiapoptotic, and antifibrotic effects.