Polysaccharide (EPS1-1) from Rhizopus nigricans might bind to TLR4 to activate the function of macrophages via MAPK and NF-κB signaling pathways. Display omitted •Polysaccharide (EPS1-1) from Rhizopus nigricans might bind to the surface receptor TLR4 to activate the function of macrophages.•EPS1-1 can activate MAPK signaling pathway to induced the activation of macrophages.•EPS1-1 can activate NF-κB signaling pathway to perform the immune activation of macrophages. Our previous reports showed that the structural features and immunologic enhancement of polysaccharide (EPS1-1) from Rhizopus nigricans. However, the molecular mechanism in cellular immunomodulatory of EPS1-1 remains unclear. Here the experiments for the molecular mechanisms of EPS1-1 on the peritoneal macrophages were performed. The results demonstrated that the expression of TLR4 was significantly improved by EPS1-1. Subsequently, the phosphorylation of p38MAPK, ERK1/2, JNK and IKKα/β were promoted. Moreover, EPS1-1 enhanced the expressions of IL-2, TNF-α and iNOS in EPS1-1-induced macrophages which were pretreated with MAPK signaling pathway inhibitors, and reduced the blocking effects of the inhibitors to the expressions of p-p38MAPK, p-ERK1/2 and p-IKKα/β. Therefore, these results illustrated that EPS1-1 could improve the immune functions of peritoneal macrophages by promoting the gene expressions of IL-2, TNF-α and iNOS via the MAPK and NF-κB signaling pathways.