Natural killer-like B (NKB) cells are a newly identified immune subset, which are separated from NK cells and B cells. NKB cells demonstrated immunoregulatory functions in elimination of microbial infection and inflammation through secretion of interleukin (IL)-12 and IL-18. However, the role of NKB cells in hepatitis B virus (HBV)-related diseases has not been reported. In this study, peripheral T cells, B cells, NK cells, and NKB cells in HBV-associated acute-on-chronic liver failure (ACLF), chronic hepatitis B (CHB), asymptomatic HBV carriers (AsC), and controls were investigated by flow cytometry. Plasma IL-12 and IL-18 levels were measured by enzyme-linked immunosorbent assay. Peripheral blood mononuclear cells from HBV-ACLF patients were stimulated with recombinant IL-12 or IL-18. Changes of immune cell percentage and nuclear factor-kappa B (NF-κB) phosphorylation were assessed. There were no statistical differences of T cell percentage, B cell percentage, or NK cell percentage among groups. NKB cell percentage within lymphocytes, and plasma IL-12 and IL-18 levels in HBV-ACLF patients were significantly elevated compared with in CHB, AsC, and controls. NKB cell percentage and IL-18, but not IL-12, had a better prognosis function for the 28-day survival status in HBV-ACLF patients. Recombinant IL-12 enhanced T cell and NK cell percentage, while only high concentrations (10 ng/mL) of IL-18 promoted NKB cell percentage in HBV-ACLF patients. High concentrations (10 ng/mL) of IL-18 induced NF-κB phosphorylation in NKB cells probably through suppression of IL-18 binding protein in HBV-ACLF patients. The current data indicated that elevated NKB cells and IL-18 might be important indicators for poor prognosis of HBV-ACLF patients. Increased IL-18 might play a positive feedback activity to NKB cells in HBV-ACLF patients.