Abstract: To explore the role of interleukin(IL)-6 in the process of liver regeneration after acetaminophen(APAP)-induced acute liver injury(ALI) throughin vitroexperiments. Methods: In vitroexperiments, AML12 cell lines which originated from normal mice hepatocytes were cultured and treated with different concentrations of APAP drugs and IL-6 neutralizing antibody(IL-6 Ab). CCK-8 method was used to detect cell viability and screen out the appropriate drug effects concentration. The IL-6 concentration was measured by ELISA. Western blot was used to detect the expression levels of liver regeneration-related proteins such as PCNA, CyclinD1, and HGF. The mRNA levels of IL-6, PCNA and CyclinD1 were measured by qRT-PCR. Datas were analyzed by GraphPad Prism 8.0 software. Results: Based on the CCK-8 data, the optimal drug concentration of 5 mmol/L APAP was selected for modeling and 0.01 μg/ml IL-6 Ab acting cells. The IL-6 concentration was 0, 1.794, 2.264, 1.658, 1.086 pg/ml at 0, 4, 12, 24, 48 h, respectively, measured by ELISA kit. Western blot results showed that compared with other time points after APAP administration on AML12 cells, the protein levels of CyclinD1 was the highest at 4 h, PCNA and HGF were the highest at 12 h(P<0.05). Compared with the 4 h APAP group, the expression of CyclinD1 and p-STAT3 protein in the APAP+IL-6 Ab group decreased(P<0.05); compared with the 12 h APAP group, the expression of PCNA and HGF in the APAP+IL-6 Ab group decreased(P<0.05). The results of qRT-PCR showed that the mRNA levels of IL-6, PCNA, and CyclinD1 in the 4 h APAP group reached the peak(P<0.05);compared with the 4 h APAP group, the PCNA and CyclinD1 levels in the APAP+IL-6 Ab group were reduced(P<0.05). Conclusion IL-6 Ab inhibits STAT3 phosphorylation by neutralizing IL-6 and subsequently inhibits liver regeneration. It is speculated that IL-6 may play a role in promoting liver regeneration after APAP-induced liver injury.