Diabetic nephropathy, a chronic and incapacitating kidney disease stemming from diabetes mellitus, poses a significant health concern. This research aimed to assess the effectiveness of nano formulated hesperidin as a potential therapeutic intervention for diabetic nephropathy, utilizing a rat model. The study delved into the intricate molecular mechanisms underlying hesperidin's impact, focusing on its modulation of the PI3K/mTOR signaling pathway. Kidney function tests, including urea and creatinine, revealed reduced levels, indicative of hesperidin's potential renoprotective effects. Western blot analysis exhibited a downregulation of PI3K and mTOR protein expression, further corroborating the therapeutic influence of nano formulated hesperidin. Concurrently, gene expression analysis via RT-PCR demonstrated a consistent reduction in PI3K and mTOR transcript levels in kidney samples from rats treated with nano formulated hesperidin. Molecular docking analysis provided additional insights, revealing a robust binding affinity between hesperidin and the PI3K/mTOR targets. This holistic approach, combining biochemical, molecular, and computational analyses, strengthens the proposition that nano formulated hesperidin holds promise as an efficacious therapeutic strategy for diabetic nephropathy. The observed reductions in kidney function markers, protein expression, and gene transcription collectively underscore the potential of nano formulated hesperidin in mitigating the deleterious effects of diabetic nephropathy, offering new avenues for targeted and effective treatment.