Lycopene (LYC) has been reported to exhibit antioxidant and immunoprotective activities, and our previous studies confirmed that LYC can alleviate multiple tissue damage induced by aflatoxin B1 (AFB1). However, it is unclear whether LYC could relieve the AFB1-induced immunosuppression. Thus, forty-eight male mice were randomly allocated and treated with LYC (5 mg kg-1) and/or AFB1 (0.75 mg kg-1) by intragastric administration for 30 days. We found that LYC alleviated AFB1-induced immunosuppression by relieving splenic structure injury and increasing the spleen weight, spleen coefficient, T lymphocyte subsets, the contents of IL-2, IFN-γ and TNF-α in serum, as well as the mRNA expression of IL-2, IFN-γ and TNF-α in spleen. Furthermore, LYC inhibited oxidative stress induced by AFB1via decreasing the levels of reactive oxygen species (ROS), hydrogen peroxide (H2O2) and malondialdehyde (MDA), while enhancing the total antioxidant capacity (T-AOC) and antioxidant enzyme activities. In addition, LYC also restrained splenic apoptosis through blocking mitochondria-mediated apoptosis in AFB1 intoxicated mice, presenting as the increase of mitochondrial membrane potential, and the decrease of cytoplasmic Cyt-c protein expression, cleaved Caspase-3 protein expression, Caspase-3/9 activities and mRNA expressions, as well as balancing the mitochondrial protein and mRNA expressions of Bax and Bcl-2. These results indicate that LYC can alleviate AFB1-induced immunosuppression by inhibiting oxidative stress and mitochondria-mediated apoptosis of mice spleen.