Oxidative stress triggered by many environmental and clinical insults results in cellular injury and death. The small GTPase rac1 promotes oxidative stress via the production of reactive oxygen species (ROS). In turn, the homeostatic response to such stress includes up‐regulation of the dual function reducing protein/DNA repair enzyme APE/redox factor‐1(ref‐1). In this report we explore the function and relationship between ref‐1 and rac1 in the setting of oxidative stress triggered by re‐oxygenation/reperfusion. In a model of mouse hepatic ischemia/reperfusion (I/R), recombinant adenoviral overexpression of ref‐1 resulted in suppression of reperfusion‐stimulated oxidative stress, NF‐κB induction, apoptosis, and acute injury, whereas down‐regulation of endogenous ref‐1 by adenoviral expression of antisense ref‐1 led to an increase in these reperfusion‐induced parameters. Ref‐1 also mitigated ROS production induced by adenoviral expression of an active form of rac1. Finally, overexpression of ref‐1 in primary hepatocytes suppressed reoxygenation‐stimulated rac1 activity. This work demonstrates a novel function of ref‐1 in inhibition of rac1 activity, and rac1‐mediated oxidative stress and injury.