Hepatitis B virus (HBV) is a major public health problem, and HBV-related acute-on-chronic liver failure (HBV-ACLF) has an extremely poor prognosis due to a lack of effective treatments. B7-H3 and B7-H4 are two novel members of the B7 superfamily that are actively involved in regulating the pathogenesis of infectious diseases. However, the intrahepatic expression of both members in HBV-ACLF patients has yet to be described. In this study, we analyzed the expression of B7-H3 and B7-H4 in HBV-ACLF biopsies by immunohistochemistry. Our results showed that both members were observed in all HBV-ACLF samples, and their expression was chiefly observed on infiltrating inflammatory cells and the damaged bile ducts. Immunofluorescence double staining showed that B7-H4 was expressed chiefly on CD3(+) T cells, CD68(+) macrophages, CK-18(+) bile ducts, and CD31(+) endothelial cells, while B7-H3 was found on all cell types detected. The expression of the programmed death (PD)-1 ligands, PD-L1 and PD-L2, was also detected in these liver tissues and they were found to be co-expressed with B7-H3 and B7-H4. These results suggest that the B7-family signaling is most likely to affect the pathogenesis of this disease, and a clear understanding of their functional roles may further elucidate the disease process.