Summary. The immune mechanism(s) that lead to hepatitis B-related acute-on-chronic liver failure (HB-ACLF) are poorly understood. Interleukin-21 is a newly discovered cytokine that is involved in autoimmune and inflammatory diseases. Its potential role in HB-ACLF remains unknown. The serum levels of 12 immune cytokines measured by cytometric bead arrays and the frequency of IL-21-secreting CD4+ T cells in peripheral blood mononuclear cells (PBMC) measured by intracellular cytokine staining were compared in moderate chronic hepatitis B (M-CHB, n = 20), severe chronic hepatitis B (S-CHB, n = 20), HB-ACLF (n = 39) and healthy controls (n = 10). PBMC from M-CHB patients or healthy subjects were stimulated with rhIL-21 in vitro, and cytokines in supernatants were measured by FlowCytomix. The frequencies of IL-21-secreting CD4+ T cells were higher in HB-ACLF (both P < 0.001) and S-CHB (P = 0.002 and 0.001) as compared to M-CHB patients and controls. Serum IL-21 levels were highest (P < 0.001) in HB-ACLF and positively associated with high MELD score (P = 0.001) and mortality (P = 0.038). Recovery from HB-ACLF was associated with reduced serum IL-21 levels (P = 0.003) and lower CD4+IL-21+ T-cell frequency (P = 0.006). The secretions of IL-1β (P < 0.001), IL-6 (P < 0.001), IL-10 (P < 0.001), IFN-γ (P = 0.001) and TNF-α (P = 0.042) from PBMC were significantly increased with rhIL-21 stimulation. In summary, IL-21 has a causal role in the development of severe liver inflammation, which is upregulated in HB-ACLF and associated with severity of liver disease.