Abstract Background Inflammatory bowel disease (IBD) associates with damage to the enteric nervous system (ENS), leading to gastrointestinal (GI) dysfunction. Oxidative stress is important for the pathophysiology of inflammation-induced enteric neuropathy and GI dysfunction. Apurinic/apyrimidinic endonuclease 1/redox factor-1 (APE1/Ref-1) is a dual functioning protein that is an essential regulator of the cellular response to oxidative stress. In this study, we aimed to determine whether an APE1/Ref-1 redox domain inhibitor, APX3330, alleviates inflammation-induced oxidative stress that leads to enteric neuropathy in the Winnie murine model of spontaneous chronic colitis. Methods Winnie mice received APX3330 or vehicle via intraperitoneal injections over 2 weeks and were compared with C57BL/6 controls. In vivo disease activity and GI transit were evaluated. Ex vivo experiments were performed to assess functional parameters of colonic motility, immune cell infiltration, and changes to the ENS. Results Targeting APE1/Ref-1 redox activity with APX3330 improved disease severity, reduced immune cell infiltration, restored GI function ,and provided neuroprotective effects to the enteric nervous system. Inhibition of APE1/Ref-1 redox signaling leading to reduced mitochondrial superoxide production, oxidative DNA damage, and translocation of high mobility group box 1 protein (HMGB1) was involved in neuroprotective effects of APX3330 in enteric neurons. Conclusions This study is the first to investigate inhibition of APE1/Ref-1’s redox activity via APX3330 in an animal model of chronic intestinal inflammation. Inhibition of the redox function of APE1/Ref-1 is a novel strategy that might lead to a possible application of APX3330 for the treatment of IBD.