Aflatoxin B<sub>1</sub> (AFB<sub>1</sub>), a threatening mycotoxin, usually provokes oxidative stress and causes hepatotoxicity in animals and humans. Luteolin (LUTN), well-known as an active phytochemical agent, acts as a strong antioxidant. This research was designed to investigate whether LUTN exerts protective effects against AFB<sub>1</sub>-induced hepatotoxicity and explore the possible molecular mechanism in mice. A total of forty-eight mice were randomly allocated following four treatment groups (<i>n</i> = 12): Group 1, physiological saline (CON). Group 2, treated with 0.75 mg/kg BW aflatoxin B<sub>1</sub> (AFB<sub>1</sub>). Group 3, treated with 50 mg/kg BW luteolin (LUTN), and Group 4, treated with 0.75 mg/kg BW aflatoxin B<sub>1</sub> + 50 mg/kg BW luteolin (AFB<sub>1</sub> + LUTN). Our findings revealed that LUTN treatment significantly alleviated growth retardation and rescued liver injury by relieving the pathological and serum biochemical alterations (ALT, AST, ALP, and GGT) under AFB<sub>1</sub> exposure. LUTN ameliorated AFB<sub>1</sub>-induced oxidative stress by scavenging ROS and MDA accumulation and boosting the capacity of the antioxidant enzyme (CAT, T-SOD, GSH-Px and T-AOC). Moreover, LUTN treatment considerably attenuates the AFB<sub>1</sub>-induced apoptosis in mouse liver, as demonstrated by declined apoptotic cells percentage, decreased Bax, Cyt-c, caspase-3 and caspase-9 transcription and protein with increased Bcl-2 expression. Notably, administration of LUTN up-regulated the Nrf2 and its associated downstream molecules (HO-1, NQO1, GCLC, SOD1) at mRNA and protein levels under AFB<sub>1</sub> exposure. Our results indicated that LUTN effectively alleviated AFB<sub>1</sub>-induced liver injury, and the underlying mechanisms were associated with the activation of the Nrf2 signaling pathway. Taken together, LUTN may serve as a potential mitigator against AFB<sub>1</sub>-induced liver injury and could be helpful for the development of novel treatment to combat liver diseases in humans a...