Overexpression of programed death-ligand 1 (PD-L1) is associated with poor prognosis in leukemia. Moreover, antitumor pharmaceuticals have been shown to induce immunoresistance, leading to reduced efficacy. Previous studies have indicated that arsenic trioxide (ATO) promotes immune evasion by inducing PD-L1 expression in solid tumors; however, little is known about its role in leukemia. A proportion of patients with acute promyelocytic leukemia were resistant to ATO therapy. Thus, this study aimed to investigate the effect of ATO on the expression of PD-L1 in leukemia cells and the underlying mechanism mediated through the nuclear factor erythroid 2 related factor (NRF2) protein. Brusatol, extracted from Brucea javanica, was selected as a unique NRF2 inhibitor, and we evaluated the possibility of using a regimen combining ATO/Brusatol in leukemia therapy. Promyelocytic NB4 and lymphocytic Jurkat cells were treated with ATO and brusatol either alone or in combination. We found that ATO significantly upregulated the expression of PD-L1 in NB4 and Jurkat cells at both the protein and mRNA levels compared with its expression in the untreated cell group. Mechanistically, ATO increased nuclear NRF2 expression and the extent of NRF2 binding to the PD-L1 promoter. Pharmacological inhibition of NRF2 by brusatol significantly blocked this effect, thereby reducing ATO-induced PD-L1 expression. In addition, the combination of brusatol and ATO showed stronger cytotoxicity than ATO alone indicated by cell counting kit-8 assay. Therefore, brusatol may further enhance the antileukemia effect of ATO not only by inhibiting ATO-induced PD-L1 expression but also by enhancing ATO-induced cytotoxicity. Our study provides a rationale for the clinical application of ATO/brusatol combination therapy.