Aflatoxin B1 (AFB1) is immunotoxic to animals and is a suspected immunosuppressant in humans. β-1,3-Glucan (BG) consists of glucose polymers and has a variety of stimulatory effects on the immune system. In this study, we investigated the role of BG on the expression of phenotypic markers and cytokine secretion in mice exposed to AFB1. We treated animals with BG (150 mg/kg, p.o., once daily) for 7 days beginning at the onset of AFB1 exposure. Exposure of animals to AFB1 alone (1250 μg/kg, p.o, once daily) for 7 days resulted in a decrease in the percentages of lymphocyte subsets (CD4+, GITR+, CD8+, TCR β+, CD3+, Foxp3+, CD4+ Foxp3+, and CD127+) as compared to an normal control (NC). However, both BG alone and BG given in conjunction with exposure to AFB1 significantly increased the percentages of these lymphocyte subsets in blood. We also observed that mice exposed to AFB1 showed reduced IL-2, TNF-α, IL-17, and IFN-γ production in the spleen and serum. In contrast, oral administration of BG alone and in conjunction with AFB1 exposure augmented the levels of these cytokines. Moreover, this finding was confirmed through RT-PCR and western blot analysis of mRNA and protein expression in the spleen. Altogether, it can be concluded from these studies that BG enhances the responses of lymphocyte subsets, including cytokine production, even when given following exposure to AFB1 immunotoxin. These data demonstrate that BG carries out its immunomodulating activity by regulating cytokine production. Our findings also provide a direction for development of specific immunomodulating therapy.