Diabetic nephropathy (DN) is one of the most common causes of chronic kidney diseases (CKDs) that have been associated with high morbidity and mortality. Recently, several studies have highlighted the protective role of vitamin D3 (VD3) and omega-3 fatty acids (O3-FAs) in CKDs. However, their effect on DN is still unclear. Aims: This study aimed to evaluate the effects of both VD3 and / or O3-FAs in slowing the progression of DN through their impact on indices of renal function, glycemic control, oxidative stress and markers of podocyte injury. Materials and methods: type I diabetes was induced in albino rats by single intraperitoneal injection of streptozotocin (65mg/kg). Rats received daily oral administration of VD3 and O3-FAs separately and in combination for 6 weeks. Results: VD3 and O3-FAs therapy improved significantly hyperglycemia, renal function tests, with concomitant decrease in total urinary protein content, urinary nephrin, a marker of podocyte injury and renal oxidative stress. However, the combined therapy was superior in its effect over VD3 and O3-FAs separately. Such results were confirmed by renal cortical tissue assessment using light microscopic examination of H&E and PAS stains in addition to transmission-electron microscopy. Conclusions: VD3 and O3-FAs have a potential beneficial effect on amelioration of structural changes in podocytes and glomerular basement membrane that was reflected as evident renal functional restoration.