Background Dietary constituents in certain bioactive substances may alleviate the toxicity of aflatoxin B1 (AFB1). Lycopene is a natural bioactive substance with powerful antioxidant, this study investigated whether dietary lycopene supplementation could alleviate AFB1-induced liver damage through inhibiting cytochrome P450 (CYP450) isozymes and improving detoxification and antioxidation with nuclear factor erythroid 2-related factor 2 (Nrf2) activation in broiler chickens. 320 healthy one-day-old male broiler chickens were randomly allotted into five groups and fed basal diet (Control), basal diet with AFB1 and 0, 100, 200 or 400 mg/kg lycopene, respectively. At 42 days of age, 8 broiler chickens per group were sampled.Results Compared to the control group, AFB1-exposed birds had depressed growth performance, hepatotoxicity and oxidative damage. The various lycopene levels prevented AFB1-induced growth depression, decreased activities and mRNA expressions of hepatic CYP450 isozymes, reduced reactive oxygen species and oxidative damage products, improved reduced glutathione (GSH)-dependent detoxification system and antioxidant system. Moreover, lycopene elevated the mRNA expressions of Nrf2 and its target genes, containing glutathione-s-transferase isozymes, GSH synthesis-related enzymes and antioxidases in AFB1-exposed broiler chickens.Conclusions As an effective anti-stress and hepatoprotective antioxidant, lycopene alleviated AFB1-induced liver damage possibly through inhibiting CYP450 isozymes and improving Nrf2-mediated detoxification and antioxidant systems in broiler chickens. Therefore, Lycopene and lycopene-enriched materials could be taken into consideration as effective hepatoprotective and anti-stress additives in poultry, and benefit the resulting food safety.