Background: Acetaminophen (APAP) overdose is a major cause of the morbidity of acute liver failure. The current clinically approved treatment for APAP poisoning, N-acetylcysteine (NAC), has a limited therapeutic window, and prolonged treatment with NAC delays liver regeneration. Mesenchymal stem cells (MSCs) also have therapeutic effects on APAP-induced mouse liver failure, but whether the effects are comparable to those of NAC has not been determined, and the mechanism still needs further exploration. Methods: Fasted C57BL/6 mice that received 500 mg/kg APAP were treated intravenously with 300 mg/kg NAC or different amounts of MSCs at 2 h after APAP to investigate survival, hepatocyte necrosis and neutrophil/macrophage recruitment. In vitro co-culture was performed to study the anti-necrotic effects of MSCs on the APAP-injured hepatocyte cell line L-O2. Results: MSCs dose-dependently rescued the C57BL/6J mice from APAP-induced liver failure, with 87.5% of MSCs (1 × 106) surviving similar to that of NAC (90%). MSC has similar effects on reduced hepatocyte necrosis and granulocytic myeloid-derived suppressor cells (MDSC) infiltration but enhanced the proportion of regenerative monocytic MDSC when compared to NAC. Mechanistically, MSCs attenuate hepatocyte necrosis by secreting hepatocyte growth factor (HGF). When HGF was knocked down, the protective effects of MSCs were reduced on APAP-induced hepatocyte necrosis and mouse liver failure. Conclusions: MSCs are comparable to NAC against APAP-induced liver failure by secreting HGF with less regenerative retardation concerns, thus facilitating the application of MSCs in clinical therapy for APAP liver failure.Keywords: Acetaminophen; Hepatocyte growth factor; Mesenchymal stem cells; N-acetylcysteine.