Recent studies indicated renal toxicity and interstitial nephritis in patients receiving leflunomide (LEFN), but the exact mechanism is still unknown. The transforming growth factor β (TGFβ)/p53/Smad2/3 pathway crucially mediates renal fibrosis. We aimed to assess the nephrotoxic effect of LEFN in mice and the possible role of TGFβ-stimulated p53/SMAD2/3 signaling. The study design involved distributing sixty male albino mice into four groups: (i) vehicle-treated mice, (ii) LEFN (2.5 mg/kg), (iii) LEFN (5 mg/kg), and (iv) LEFN (10 mg/kg). The drug was given orally every 48 h and continued for 8 weeks. Blood samples were then taken from mice for the determination of kidney function parameters. Right kidneys were used for histopathologic staining and immunohistochemistry, whereas left kidneys were frozen and used for Western blot analysis of the target proteins, p-p53 and Smad2/3. Results indicated that chronic administration of LEFN in mice resulted in a four- and nine-fold increase in serum urea and creatinine levels, respectively. Kidney specimens stained with hematoxylin and eosin or periodic acid–Schiff showed significant histopathological manifestations, such as cellular irregularity, interstitial congestion, and moderate lymphocytic inflammatory infiltrate in mice treated with LEFN. Western blotting indicated upregulation of the p-p53/Smad2/3 proteins. LEFN, especially in the highest dose (10 mg/kg), produced prominent nephrotoxicity in mice. This toxicity is mediated through stimulating fibrotic changes through TGFβ-stimulated p53/Smad2/3 signaling and induction of glomerular and tubular apoptosis. An improved understanding of LEFN-induced nephrotoxicity would have great implications in the prediction, prevention, and management of leflunomide-treated rheumatic patients, and may warrant further clinical studies for following up these toxidromes.