AbstractChemoresistance is the main obstacle in osteosarcoma (OS) treatment; however, the underlying mechanism remains unclear. In this study, it is discovered that DDRGK domain‐containing protein 1 (DDRGK1) plays a fundamental role in chemoresistance induced in OS. Bioinformatic and tissue analyses indicate that higher expression of DDRGK1 correlates with advanced tumor stage and poor clinical prognosis of OS. Quantitative proteomic analyses suggest that DDRGK1 plays a critical role in mitochondrial oxidative phosphorylation. DDRGK1 knockout trigger the accumulation of reactive oxygen species (ROS) and attenuate the stability of nuclear factor erythroid‐2‐related factor 2 (NRF2), a major antioxidant response element. Furthermore, DDRGK1 inhibits ubiquitin‐proteasome‐mediated degradation of NRF2 via competitive binding to the Kelch‐like ECH‐associated protein 1 (KEAP1) protein, which recruits NRF2 to CULLIN(CUL3). DDRGK1 knockout attenuates NRF2 stability, contributing to ROS accumulation, which promotes apoptosis and enhanced chemosensitivity to doxorubicin (DOX) and etoposide in cancer cells. Indeed, DDRGK1 knockout significantly enhances osteosarcoma chemosensitivity to DOX in vivo. The combination of DDRGK1 knockdown and DOX treatment provides a promising new avenue for the effective treatment of OS.