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Adipose-derived stromal vascular fraction (SVF) cells, a rich source of primary stem/stromal cells, are promising for administering cell therapy for patients with acute-on-chronic liver failure (ACLF). We evaluated the therapeutic effects of CD34+/CD34− SVF cells in hepatocyte cotransplantation in a rat model of ACLF.ACLF was induced in Sprague–Dawley rats by temporary bile duct ligation (chronic) and D-galactosamine administration (acute). Donor hepatocytes and SVF cells were isolated from Tg(UBC–emGFP) rats and human adipose tissues, respectively. Sorted CD34+ and CD34− SVF cells were separated using a magnetic bead system. Rat hepatocytes and SVF cells (labeled with the PKH26 fluorescent dye) were intraportally transplanted into ACLF rats. The surviving animals were sacrificed at 1 and 2 weeks after the transplantation.ACLF was evidenced by the onset of acute coagulopathy and hepatocyte necrosis in fibrotic livers. Compared with the control group, the unsorted group showed less biliary ductular proliferation and fibrosis at 1 week after the transplantation. Furthermore, prominent biliary ductular proliferation and significantly progression of fibrosis (P = .005) were observed in the CD34− group than in the CD34+ group. At 2 weeks, the serum level of alkaline phosphatase was significantly higher in the CD34− group than in the CD34+ group (P = .007). The transplanted SVF cells were found in the periportal regions at 1 week, whereas donor hepatocytes were not detected and proliferation of endogenous hepatocytes was observed at times.Compared with CD34− SVF cells, cotransplantation of CD34+ SVF cells resulted in the early amelioration of liver fibrosis and biliary ductular proliferation. Heterogeneous therapeutic effects of SVF cells were associated with CD34 positivity, which poses translational significance in future cell therapy.