To evaluate the protective effect of Zhizi Huangqi Shanzha formula (, ZHSF) on aflatoxin-induced liver injury.The protective effect of ZHSF on the aflatoxin-induced liver injury was evaluated by histological observation, blood cell analysis, evaluation of liver function and immunity, and gut microbiota analysis.ZHSF can significantly up-regulate the percentage of lymphocytes and eosinophils in the blood of Aflatoxin B1-intoxicated mice, down-regulate the levels of serum aspartate aminotransferase, alanine aminotransferase, and malondialdehyde, and recover the liver tissue structure. Aflatoxin poisoning induces a variation of the intestinal flora of mice, and ZHSF may recover the variation of intestinal flora induced by Aflatoxin B1. Cluster analysis showed that the intestinal flora of mice in the intervention group was more similar to that of the control group. Correlation analysis showed that Lachnospiraceae, Desulfovibrio, and Lactobacillus may be the key flora for the pharmacological effects of ZHSF.ZHSF may protect against aflatoxin-induced liver damage, improve immunity, and inhibit oxidative stress by regulating the composition and relative abundance of intestinal flora, which makes it a promising liver-protective candidate drug.