Objective · To evaluate protective effects of recombinant human IL-1Ra (rhIL-1Ra) on acute liver injury in vitrousing D-galactosamine (D-GalN) and HepG2 cells to establish the D-galactosamine (D-GalN)-induced HepG2 cells injury models. Methods · Models of HepG2 cells injury inducedD-GalN was established. HepG2 cells were maintained in mediums which contained different concentration of D-GalN (0.02, 0.2, 2 or 4 mg/mL) for different time (1, 2 or 3 d). Optimized concentration and time of D-GalN were used to analyze cell viability and morphology. A serial dose of rhIL-1Ra (10, 20 or 50 μg/mL) was used to treat HepG2 cells which were challenged with D-GalN. Cell apoptosis and the levels of intracellular reactive oxygen species (ROS) were analyzed in different treatment groups. Real-time PCR was employed to analyze the mRNA levels of IL-1β, IL-6 and TNF-α in cells. ERK1/2 inhibitor (SCH772984) was used to confirm whether ERK1/2 phosphorylation played a critical role in IL-1Ra protecting hepatocytes or not. Results · Cell viability was significantly decreasedD-GalN whose concentration was 4 mg/mL in HepG2 cells after 2 d. Compared with the control group, rhIL-1Ra could significantly improve cell survival and down-regulate the level of ROS in the cells. RhIL-Ra also could suppress of pro-apoptotic cytokines factors (IL-1β, IL-6 and TNF-α) inducedD-GalN in HepG2 cells. The results also showed that erk1/2 signaling pathways have certain effect on mediating the injury of rhIL-1Ra to protect hepatocytes. Conclusion · RhIL-1Ra can protect hepatocytes toxinsdirectly targeting hepatocytes and inhibit cells apoptosisactivating ERK1/2 pathway in HepG2 cells.