Simple SummaryBladder cancer is one of the most common malignant tumors in the urinary system. Pirarubicin (THP) perfusion therapy is an important treatment method for non-muscle-invasive bladder cancer. However, an increasing number of cases of recurrence or progression of bladder cancer caused by THP resistance have been reported. Therefore, we urgently need to find the critical genes that cause THP resistance in bladder cancer. In this study, we obtained the critical gene AKR1C1 that causes resistance of bladder cancer to THP via CRISPR/dCas9 SAM. This study not only verified that AKR1C1 can cause resistance of bladder cancer cells to THP both in in vivo and in vitro, but also found that THP treatment can gradually increase the expression of AKR1C1, thus causing resistance to the drug. In addition, we also found that aspirin, the AKR1C1 inhibitor, and tempol, the ROS scavenger, can effectively overcome the drug resistance caused by AKR1C1.AbstractNon-muscle-invasive bladder cancer (NMIBC) is a common tumor of the urinary system. Given its high rates of recurrence, progression, and drug resistance, NMIBC seriously affects the quality of life and limits the survival time of patients. Pirarubicin (THP) is a bladder infusion chemotherapy drug recommended by the guidelines for NMIBC. Although the widespread use of THP reduces the recurrence rate of NMIBC, 10–50% of patients still suffer from tumor recurrence, which is closely related to tumor resistance to chemotherapy drugs. This study was performed to screen the critical genes causing THP resistance in bladder cancer cell lines by using the CRISPR/dCas9-SAM system. Thus, AKR1C1 was screened. Results showed that the high expression of AKR1C1 could enhance the drug resistance of bladder cancer to THP both in vivo and in vitro. This gene could reduce the levels of 4-hydroxynonenal and reactive oxygen species (ROS) and resist THP-induced apoptosis. However, AKR1C1 did not affect the proliferation, invasion, or migration of the bladder cancer cells. Aspirin, which is an AKR1C1 inhibitor, could help reduce the drug resistance caused by AKR1C1. After receiving THP treatment, the bladder cancer cell lines could upregulate the expression of the AKR1C1 gene through the ROS/KEAP1/NRF2 pathway, leading to resistance to THP treatment. Using tempol, which is an inhibitor of ROS, could prevent the upregulation of AKR1C1 expression.