The transcription factor nuclear factor (erythroid-derived 2)-like 2 (NRF2) is one of the master regulators that control hundreds of genes containing antioxidant response elements (AREs). The NRF2-ARE pathway plays a complex role in colorectal cancer (CRC). NRF2 activity is known to be regulated by KEAP1-CUL3 E3 ligase-mediated ubiquitination, indicating the importance of deubiquitination regulation. However, the deubiquitinase (DUB) of NRF2 remains unknown. Here, by screening a DUB library, we identified DUB3 as a DUB that remarkably stabilized NRF2. Further experiments demonstrated that DUB3 promoted NRF2 stability and transcriptional activity by decreasing the K48-linked ubiquitination of NRF2. Coimmunoprecipitation studies revealed interactions between NRF2 and DUB3, as well as between KEAP1 and DUB3, indicating that NRF2, DUB3, and KEAP1 formed a large functional complex. Importantly, ectopic expression of DUB3 caused NRF2-dependent chemotherapy resistance in colon cancer cell lines. Thus, to the best of our knowledge, our findings are the first to identify DUB3 as a NRF2 DUB and may provide a new strategy against chemotherapy resistance in CRC and other NRF2-related diseases.