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The purpose of this study was to test the protective effect of Withania somnifera (WS) against the harmful effects of mercuric chloride (HgCl2)-induced kidney failure at the histological, biochemical, and immune levels in Wistar rats. The study assessed the biochemical and immunological changes in five groups (n = 6): Group 1 (G1) was the negative control, and the other rats received a single subcutaneous dose of HgCl2 (2.5 mg/kg in 0.5 mL of 0.9% saline solution) and randomly divided into 4 groups. Group 2 (G2) was the positive control and left without treatment. Groups 3, 4, and 5 (G3, G4, and G5) were treated with different doses of WS root powder for 30 days. The HgCl2-positive group showed significant signs of renal toxicity as reflected by increased levels of kidney function parameters (blood urea nitrogen, urea, and creatinine), inflammatory biomarkers, immunological indices (SDF-1, IL-6, NGAL, and KIM-1), and oxidative stress (SOD, TAC, CAT, GSH, and MDA). The positive group rats also showed drastic pathological changes in renal tissues. Different doses of WS treatment significantly reduced the levels of all biochemical markers and decreased pathological damage to the kidney tissues. The antioxidant, phenolic, and flavonoid constituents of WS root powder helped protect rats’ kidneys against HgCl2-induced kidney toxicity in male rats.