Objective To explore the therapeutic effects of soluble cytokines secreted by mesenchymal stem cells (MSCs) on acute liver failure (ALF). Methods MSCs isolated from Sprague-Dawley rats were determined by FACS analysis. Conditioned medium derived from MSCs (MSCs-CM) was collected and analyzed by a cytokine microarray. SD rats were divided into 3 groups: (1) ALF + dulbecco's modified eagle medium (DMEM) group: 1 ml DMEM was injected into SD rats after D-Gal administration; (2) ALF + MSCs group: 1 ml MSCs (1×106) was injected into SD rats after D-Gal administration; (3) ALF + MSCs-CM group: 1 ml MSCs-CM was injected into SD rats after D-Gal administration. Biochemical indicators, survival rate, histology and inflammatory factors were studied. Exogenous recombinant rat IL-10, anti-rat IL-10 antibody and AG490 (STAT3 signaling pathway inhibitor) were administrated to explore the therapeutic mechanism of MSCs-CM. Results The respective serum biochemical indexes of ALF + DMEM group, ALF + MSCs group, and ALF + MSCs-CM group were: ALT (1 709.8 ± 372.1, 865.5 ± 52.8, 964.7 ± 414.6 U/L), AST (4 234.0 ± 807.3, 2 440.8 ± 511.9, 2 739.8 ± 587.3 U/L), and TBil (79.3 ± 10.9, 43.8 ± 7.0, 61.2 ± 6.7 μg/L). The survival rates of the three groups were 10.0%, 80.0%, and 70.0%, respectively. The levels of inflammatory factors in each group were IFN-γ (69.8 ± 4.7, 46.4 ± 4.3, 54.6 ± 2.4 pg/ml), IL-1β (58.5 ± 7.6, 40.5 ± 6.9, 44.1 ± 6.0 pg/ml), IL-6 (71.9 ± 16.1, 38.4 ± 7.7, 45.3 ± 9.0), and IL-10 (38.3 ± 6.0, 75.4 ± 11.1, 59.6 ± 11.9 pg/ml). Protein microarray results suggested that MSCs-CM expresses a variety of inflammatory-related cytokines, with IL-10 levels being most pronounced. IL-10 (ALT 1 126.9 ± 419.3 U/L, AST 2 370.8 ± 561.2 U/L) alone significantly reduced transaminase levels compared with ALF group (ALT 1 709.8 ± 372.1 U/L, AST 4 234.0 ± 807.3 U/L), while anti-IL-10 antibody (ALT 1 568.5 ± 325.4 U/L, AST 4 043.7 ± 819.0 U/L) neutralized the therapeutic effect of MSCs-CM (ALT 964.7 ± 414.6 U/L, AST 2 739.8 ± 587.3 U/L). IL-10 could significantly increase the level of pSTAT3 in ALF rats (0.93 ± 0.03 vs 0.68 ± 0.01), while STAT3 inhibitor AG490 (0.84 ± 0.04) could decrease the expression of pSTAT3 and reverse the therapeutic effect of IL-10. Conclusion The factors released by MSCs, especially IL-10, have the potential therapeutic effect on ALF, and STAT3 signaling pathway may mediate the anti-inflammation effects of IL-10. Key words: Mesenchymal stem cell; Acute liver failure; Conditioned medium; Immunoregulation; STAT3 signaling pathway