Background: Ulcerative colitis (UC) is a complex, chronic, and relapsing inflammatory disorder categorized by chronic inflammation followed by colonic damage. D-Pinitol has been recognized for its numerous pharmacological properties, counting antioxidant, antiulcer, and anti-inflammatory potential. Aim: The aim of the study was to measure the plausible mechanisms of action of pinitol on an experimental model of (2, 4, 6-trinitrobenzene sulfonic acid [TNBS])-induced UC in rats. Materials and Methods: TNBS (100 mg/kg, in 50% ethanol) was employed to induce UC in overnight fasted Sprague–Dawley rats. The rats have received either vehicle or (5-aminosalicylic acid [5-ASA]) or pinitol (5 or 10 or 20 mg/kg), p.o. for 14 days. Innumerable biochemical and molecular analysis were achieved in colon tissue. Results: Rectal instillation of TNBS resulted in the induction of colonic damage reproduced by marked (P < 0.05) reduced in colonic total antioxidant capacity (TOC) and significant (P < 0.05) surge in colonic oxido-nitrosative, myeloperoxidase, and hydroxyproline levels. However, administration of pinitol (10 and 20 mg/kg) effectively inhibited these TNBS-induced colonic damages. Real-time polymerase chain reaction (PCR) analysis recommended that TNBS-induced upregulated cytokine (Tumour necrosis factor-α [TNF-α], (interleukins)-1 [ILs] β, and IL-6), and nuclear factor-κB (NF-κB) messenger ribonucleic acid expressions were effectively (P < 0.05) condensed by pinitol. Western blot analysis recommended that pinitol conspicuously (P < 0.05) augmented tight junction proteins (claudin-1, occludin, and Zonula occludens-1 (ZO-1)) expression to improve colon functions. TNBS-induced histopathology modification in the colon was significantly (P < 0.05) diminished by pinitol. Conclusion: D-Pinitol ameliorated TNBS-induced UC through inhibition of raised oxidative stress (TOC, superoxide dismutase, glutathione, and Malondialdehyde) and inflammatory release (TNF-α, ILs, and NF-κB), which further progresses the intestinal barrier through activation of colonic tight junction proteins (ZO-1, claudin-1, and occludin).