Oxidative stress is an important molecular mechanism for kidney injury in aflatoxin B1 (AFB1) nephrotoxicity. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a master transcription factor for regulating the cellular oxidative stress response, which has been confirmed in animal models. Lycopene (LYC), a natural carotenoid, has received extensive attention due to its antioxidant effect with the activation of Nrf2. However, the role of LYC in protecting against AFB1-induced renal injury is unknown. To evaluate the chemoprotective effect of LYC on AFB1-induced renal injury, forty-eight male mice were randomly divided into 4 groups and treated with LYC (5 mg per kg of bodyweight) and/or AFB1 (0.75 mg per kg of bodyweight) by intragastric administration for 30 days. AFB1 and LYC were respectively dissolved in olive oil. We found that AFB1 exposure significantly increased the serum concentrations of blood urea nitrogen (BUN) and serum creatinine (SCR), and caused damage to the renal structure. Notably, LYC potentially alleviated AFB1-induced kidney lesions through attenuating AFB1-induced oxidative stress. Renal nuclear factor-erythroid 2-related factor 2 (Nrf2) and its downstream target gene (CAT, NQO1, SOD1, GSS, GCLM and GCLC) translation and protein expression were ameliorated by pretreatment with LYC in AFB1-exposed mice. These results suggested that LYC potentially alleviates AFB1-induced renal injury. This effect may be attributed to the enhancement of renal antioxidant capacity with the activation of the Nrf2 antioxidant signaling pathway.