Abstract Background Radiotherapy (RT) is one of the major therapeutic approaches to hepatocellular carcinoma (HCC). Ionizing radiation (IR) inducing the generation of reactive oxygen species (ROS) leads to a promising antitumor effect. However, the dysregulation of the redox system often causes radioresistance and impairs the efficacy of RT. Increasing evidence indicates that nuclear protein 1 (NUPR1) plays a critical role in redox reactions. In this study, we aim to explore the role of NUPR1 in maintaining ROS homeostasis and radioresistance in HCC. Methods The radioresistant role of NUPR1 was determined by colony formation assay, comet assay in vitro, and xenograft tumor models in vivo. Probes for ROS, apoptosis assay, and lipid peroxidation assay were used to investigate the functional effect of NUPR1 on ROS homeostasis and oxidative stress. RNA sequencing and co-immunoprecipitation assay were performed to clarify the mechanism of NUPR1 inhibiting the AhR/CYP signal axis. Finally, we analyzed clinical specimens to assess the predictive value of NUPR1 and AhR in the radiotherapeutic efficacy of HCC. Results We demonstrated that NUPR1 was upregulated in HCC tissues and verified that NUPR1 increased the radioresistance of HCC in vitro and in vivo. NUPR1 alleviated the generation of ROS and suppressed oxidative stress, including apoptosis and lipid peroxidation by downregulating cytochrome P450 (CYP) upon IR. ROS scavenger N-acetyl-L-cysteine (NAC) and CYP inhibitor alizarin restored the viability of NUPR1-knockdown cells during IR. Mechanistically, the interaction between NUPR1 and aryl hydrocarbon receptor (AhR) promoted the degradation and decreased nuclear translation of AhR via the autophagy-lysosome pathway, followed by being incapable of CYP’s transcription. Furthermore, genetically and pharmacologically activating AhR abrogated the radioresistant role of NUPR1. Clinical data suggested that NUPR1 and AhR could serve as novel biomarkers for predicting the radiation response of HCC. Conclusions Our findings revealed the role of NUPR1 in regulating ROS homeostasis and oxidative stress via the AhR/CYP signal axis upon IR. Strategies targeting the NUPR1/AhR/CYP pathway may have important clinical applications for improving the radiotherapeutic efficacy of HCC.