Hepatic ischemia/reperfusion (I/R) injury is characterized by the generation of reactive oxygen species (ROS), such as superoxide anions and hydrogen peroxide. The aim of this study is to investigate whether antioxidative gene delivery by our polylipid nanoparticles (PLNP) is an effective approach for prevention of the injury. Polyplexes of extracellular superoxide dismutase (EC‐SOD) and/or catalase genes were injected via the portal vein 1 day prior to a warm I/R procedure in mice. The effects of the gene delivery were determined 6 hours after starting reperfusion. PLNP‐mediated antioxidative gene delivery led to a marked increase in human EC‐SOD and catalase gene expression in the liver. Liver superoxide dismutase (SOD) and catalase activity both increased approximately 10‐fold. Increased liver superoxide anion levels caused by the I/R procedure were reduced to normal levels by EC‐SOD gene delivery. The overexpression of these 2 antioxidative genes significantly suppressed the I/R‐induced elevation of serum alanine aminotransferase (ALT) levels, decreased liver malondialdehyde content, restored glutathione reserve, and improved liver histology. In conclusion, EC‐SOD or catalase gene delivery by PLNP resulted in high levels of the transgene activity in the liver, and markedly attenuated hepatic I/R injury. The protection is directly associated with elevated antioxidative enzyme activity as the result of the gene delivery. This novel approach may become a potential therapy to improve graft function and survival after liver transplantation. Liver Transpl 12:1869–1879, 2006. © 2006 AASLD.