Diabetic nephropathy is recognized as the predominant cause of end-stage renal disease worldwide. In reaction to metabolic stress, the peptide hormone spexin-14, is synthesized in both central and peripheral tissues. Its level is reduced in type II diabetes mellites and may play a role in glucose metabolism. However, in the context of DN, the mechanisms through which spexin exerts its effects remain largely unknown. This research employed a rat model of DN to explore the therapeutic potential and the underlying mechanisms associated with spexin treatment. For the development of this experimental model, rats were subjected to an eight-week regimen of a high-fat, high-fructose diet prior to receiving a single dose of streptozotocin (35 mg/kg body weight). Subsequently, spexin was administered subcutaneously on a daily basis for a duration of eight weeks at a dosage of 50 μg/kg body weight. The evaluation methods employed encompassed renal function assessments, macromorphological examinations, histopathological evaluations, and analyses of inflammatory and oxidative stress mediators. Additionally, immunohistochemical staining for NF-kB and E-cadherin, along with PCR analysis of mTOR, Bcl2, and Bax gene expressions in renal tissues, were conducted. Following the administration of spexin to the diabetic rats, there was a significant reduction in serum levels of glucose, urea, creatinine, and inflammatory cytokines (IL-1β, TNF-α), alongside a marked restoration of antioxidant enzyme activities. Furthermore, a significant decline in the levels of NF-κB, mTOR, and Bax was noted and accompanied with increased expressions of Bcl-2 and E-cadherin proteins. The observed improvements in histopathological changes significantly corroborated the biochemical results. In summary, spexin has proven to be effective in alleviating DN by its capacity to mitigate metabolic disturbances, oxidative stress, inflammation, and apoptosis.