In this study, we investigated the protective efficacy of extracellular polysaccharide from Cordyceps militaris (CEP-I) in liver and kidney and their regulating effect on gut microbiota against Pb-induced toxicity in vivo. The results indicated that CEP-I could reduce the Pb2+ content and organ index of liver and kidney in mice. Besides, biochemical analysis showed that CEP-I could improve the activity of glutathione peroxidase (GSH-Px), malondialdehyde (MDA) and superoxide dismutase (SOD) in serum and organs, restore the physiological indexes of total protein (TP), albumin (ALB), blood urea nitrogen (BUN) and creatinine (CRE) in serum and decrease the enzyme activity of lactate dehydrogenase (LDH) and aspartate aminotransferase (AST) in the liver and kidney of mice poisoned by Pb2+. This indicated that CEP-I has a protective effect on organs against damage in mice. In addition, CEP-I could regulate the expression of key proteins in the Nrf2 signaling pathway, including NF-E2-related factor 2 (Nrf2), Kelch-like ECH-associated protein-1 (Keap1), Heme oxygenase (HO-1) and NAD(P)H: quinone oxidoreductase 1 (NQO1). Furthermore, the intestinal flora analysis results indicated that CEP-I also has the capacity to regulate the intestinal flora imbalance caused by Pb2+ in poisoned mice. In conclusion, we hope that this study can provide theoretical basis for the treatment of tissue damage induced by Pb2+.