Serine protease inhibitors (serpins) are involved in inflammation, coagulation, fibrinolysis, tumor suppression, molecular chaperone, chromatin densification, and hormone transport. However, their anti-intoxication activity has not been determined. Here, we heterologously expressed the serpin gene from Lentinula edodes in Escherichia coli and purified the recombinant serpin protein from L. edodes (rLeSPI). Then, we administered alcohol and active protein or Haiwangjinzun as a positive control to mice via gavage to evaluate the anti-intoxication activities of rLeSPI in vivo. We also investigated the protective effects of rLeSPI on acute alcohol-induced liver injury in mice by physiological and biochemical assays. The assay results for the anti-intoxication activity revealed that pretreating mice with 5 mg/kg rLeSPI for 0.5 h before gavage with Erguotou liquor (56%V EtOH, 0.15 ml/10 g) significantly prolonged the tolerance time and shortened the intoxication time relative to the results of the control group, thereby proving its anti-intoxication activities. The biochemical analysis showed that rLeSPI improved glutathione peroxidase activity, which was evidently reduced by ethanol. Additionally, rLeSPI significantly improved the activity of aldehyde dehydrogenase, which is important in alcohol metabolism, and reduced the intracellular malondialdehyde content, aspartate amino transferase, and alanine amino transferase activity. We concluded that LeSPI displayed anti-intoxication activity and exerted protective effects against acute alcohol-induced liver injury, providing new insight into the prevention of alcoholism and alcohol-related diseases.