Interleukin (IL)-38 is a poorly characterized cytokine of the IL-1 family with anti-inflammatory activity. The role of IL-38 in liver injury remains unknown. We have investigated the potential effect of hydrodynamic-based gene delivery to express human IL-38 in mice with concanavalin A (Con A)-induced liver injury. Transfer of plasmid DNA encoding IL-38 significantly reduced hepatic toxicity and serum levels of aspartate aminotransferase and alanine aminotransferase compared with administration of a control plasmid. Moreover, IL-38 expression dramatically reduced serum levels of several pro-inflammatory cytokines, such as tumor necrosis factor-α, interferon-γ, IL-6, IL-17, and IL-22, but not levels of the anti-inflammatory cytokine IL-10. These results suggest that in vivo expression of human IL-38 in mice has hepatoprotective effects against Con A-induced liver injury by inhibition of inflammatory cytokine production.