Objective To investigate the effect of total flavonoids from mulberry bark (TFMB) on acute liver injury (ALI) by antagonizing UⅡ/UT system and regulating JAK2/STAT3 signaling pathway. Methods Rats were randomly divided into control group, ALI model group, MgIG group, Urantide group and TFMB group. MgIG and Urantide were intravenously injected at a dose of 10 mg . kg^-1 in MgIG group and Urantide group, respectively. TFMB groups was gavaged with 1.8, 3.6 and 7.2 g . kg^-1, respectively; the control group and ALI model group were given the same amount of normal saline by gavage, and the treatment cycle of each group was continuous for 14 days. Results The ALI model of rats was reproduced with CCl₄, and the pathological manifestations were obvious liver edema, severe steatosis, hepatic Sinus Dilation, and the expression of EIF4EBP1 and p-p70s6 protein increased. At the same time, UⅡ/UT system and JAK2/STAT3 pathway were activated, and p-JAK2 and p-STAT3 were mainly expressed in the injured site. After preventive intervention with tfmb, compared with ALI model group, the pathological symptoms of liver were significantly reduced, the expression of p-P70s6 and EIF4EBP1 were significantly reduced, and the activation of UⅡ/UT system and JAK2/STAT3 pathway were inhibited. In addition, in the H₂O₂ induced rat hepatocyte (BRL3A) cell model, TFMB also inhibited JAK2/STAT3 pathway and proliferation. Conclusion TFMB may play a preventive and therapeutic role in CCl₄ induced acute liver injury in rats by antagonizing UⅡ/UT system and regulating JAK2/STAT3 signaling pathway.