Dipeptidyl peptidase-4 inhibitor (DPP-4 inhibitor) such as sitagliptin has been presented as antidiabetic drugs and has numerous restorative advantages over different diseases; however, its defensive role against aflatoxin b1 (AFB1) liver toxicity has not been previously examined. Wistar rats (65 weeks, male) were utilized in the investigation. Animals were divided into five different groups (n = 10): control; AFB1; AFB1 + Sita (50); AFB1 + Sita (100); and Sita (100). Sitagliptin significantly (*p ≤ .05, **p ≤ .01, and ***p ≤ .001) altered the levels of various serum liver enzymes (lactate dehydrogenase, alkaline phosphate, aspartate aminotransferase, and alanine aminotransferase). It decreased the concentration of an oxidative stress marker, that is, malondialdehyde and increased the level of antioxidant enzymes such as reduced glutathione, catalase, superoxide dismutase, and glutathione peroxidase in AFB1-administered rats. It also improved the Nrf2 expression and HO-1 level in AFB1-intoxicated rats. This investigation discusses innovative evidence on the protective role of sitagliptin against AFB1-induced hepatotoxicity in rats.