AbstractAimAcute‐on‐chronic liver failure (ACLF), a disease with poor prognosis, is reportedly caused by cellular senescence due to mitochondrial dysfunction. In this study, we described and analyzed the underlying mechanism of a novel approach for ACLF using ABT263/navitoclax (Navi) that selectively eliminates senescent cells.MethodsIrradiation‐induced senescent hepatocytes were used for in vitro evaluation of the effects of Navi on ACLF (n = 6 for each group). Lipopolysaccharide‐ and carbon tetrachloride‐induced ACLF mouse model was used for in vivo evaluation of the effects of Navi administration compared with the control using one‐way or two‐way analysis of variance, followed by Student's t‐test or Kruskal–Wallis test. The effects on the senescence‐associated secretory phenotype (n = 8 for each group) and mitochondrial functions, including adenosine triphosphate concentration and membrane potential (n = 8 for each group), were investigated using real‐time polymerase chain reaction, immunohistochemistry, and enzyme analysis.ResultsNavi eliminated irradiation‐induced senescent hepatocytes in vitro, leading to non‐senescent hepatocyte proliferation. Navi eliminated senescent cells in the liver in vivo, resulting in downregulation of mRNA expression of senescence‐associated secretory phenotype factors, a decrease of liver enzymes, and upregulated proliferation of non‐senescent cells in the liver. Regarding mitochondrial functional assessment in the liver, adenosine triphosphate concentration and membrane potential were upregulated after Navi administration in vitro and in vivo.ConclusionsNavi may ameliorate ACLF damage by eliminating senescent cells in the liver, downregulating senescence‐associated secretory phenotype factors, and upregulating mitochondrial functions. We believe that this novel approach using Navi will pave the way for ACLF treatment.