Background: NSC697923, a ubiquitin-conjugating enzyme E2 (UBE2) inhibitor, was suggested as an agent to degrade hypoxia-inducible factor 1 alpha subunit (HIF1 alpha), a key factor in radiation resistance. We attempted to clarify whether NSC697923 could overcome radiation resistance. Materials and Methods: Radiation resistance and expression of HIFs were evaluated in radiation-sensitive HCT116 and -resistant SW480 cells treated with or without NSC697923 and radiation under normoxia and hypoxia in vitro and in vivo. We examined NSC697923-regulated genes using RNA sequencing. Results: HIF expression significantly increased under hypoxia with an increase of cellular radiation resistance in vitro and in vivo. The therapeutic activity of NSC697923 was higher in radiation-resistant SW480 than radiation-sensitive HCT116 in vivo. Next-generation RNA sequencing revealed that NSC697923 regulated the expression of cell migration-inducing protein, hyaluronan binding (CEMIP) and apelin (APLN) genes, that are related to HIF pathways. Conclusion: NSC697923 might effectively regulate HIF families, and be a promising partner with radiation to overcome resistance.