〔Abstract〕 Objective To investigate the role of Toll-like receptor 4(TLR4) in hepatocyte regeneration after acetaminophen(APAP)-induced injury in human normal liver cell(L02) and its possible mechanism. Methods L02 cells were culturedin vitro, and cell viability was detected by CCK-8 assay. The optimal concentration and duration of APAP and the concentration of TLR4 inhibitor(TAK-242) were determined. The protein expression levels of nuclear factor-κB(NF-κB), microtubule-associated protein light chain 3(LC3), p62, receptor interacting protein kinase 1(RIP1), receptor interacting protein kinase 3(RIP3), signal transducer and activator of transcription 3(STAT3), phosphorylation of STAT3(p-STAT3), proliferating cell nuclear antigen(PCNA) and Cyclin D1 were detected by Western blot. The mRNA expression levels of TLR4, NF-κB, tumor necrosis factor-α(TNF-α), interleukin-6(IL-6), interleukin-1β(IL-1β), PCNA, Cyclin D1 and Ki67 were detected by qRT-PCR. Results According to the results of CCK-8, L02 cells were treated with 5 mmol/L APAP for 24, 36, 48 h to simulate liver injury and regeneration modelin vitro, and TAK-242 100 nmol/L was pretreated 2 h before APAP to inhibit TLR4. Compared with the control group, the protein levels of NF-κB, RIP1, p-STAT3, PCNA, Cyclin D1 and the mRNA levels of TNF-α, IL-1β and PCNA increased in the APAP 24 h group; the protein levels of NF-κB, RIP1, RIP3, p-STAT3, PCNA, Cyclin D1 and the mRNA levels of TLR4, NF-κB, TNF-α, IL-1β, PCNA and Cyclin D1 increased in the APAP 36 h group; the protein levels of NF-κB, RIP1, p-STAT3, PCNA, Cyclin D1 and the mRNA levels of TLR4, NF-κB, TNF-α, IL-1β, IL-6, PCNA, Cyclin D1 and Ki67 increased in the APAP 48 h group. The protein levels of NF-κB, RIP1, RIP3, p-STAT3, PCNA, Cyclin D1 and the mRNA levels of TLR4, NF-κB, TNF-α, IL-1β, IL-6, PCNA, Cyclin D1, Ki67 significantly decreased in APAP+TAK-242 24 h and 48 h group than the APAP group at the same time point; the protein levels of NF-κB, PCNA and the mRNA levels of TLR4, NF-κB, TNF-α, IL-1β, IL-6, PCNA and Ki67 in APAP+TAK-242 36 h group were also significantly lower than those in APAP 36 h group. Compared with the control group, autophagy was activated in the APAP group, while autophagy was inhibited in the APAP+TAK-242 group. Conclusion TLR4 may affect the TLR4/NF-κB pathway, up-regulate the levels of inflammatory factors and autophagy, and promote hepatocyte regeneration after APAP-induced liver injury in L02 cells.