Nucleophosmin/B23 (NPM) is a nuclear protein with prosurvival and ribosomal RNA processing functions. However, the potential role of NPM involved in drug-resistance in leukemia has not been investigated clearly. In this study, we generated an adriamycin (ADM)-resistant lymphoblastic cell line Molt-4/ADR (MAR) by stepwise induction. Cell proliferation, sensitivity to chemotherapy agents and expressions of drug resistance related molecules were assessed. The IC50 of Molt-4 cells were 0.58±0.11μmol/L and MAR cells were 22.56±1.94μmol/L, meaning MAR cells were 38.63 fold resistant to Molt-4 cells. Furthermore, MAR cells gained an expression of mdr-1 (P-gp) and a higher expression of NPM compared to Molt-4 cells. Knockdown of NPM by RNA interference (RNAi) suppressed the viability of both Molt-4 and MAR cells. After NPM RNAi, the IC50 of MAR and Molt-4 cells were 3.83±0.38μmol/L and 0.19±0.02μmol/L respectively. Both of them revealed an increase of drug sensitivity with down-regulation of mdr-1 and Akt/mTOR signaling. Knockdown of mdr-1 could also reverse the drug resistance, with no change in NPM expression. It could be concluded that knockdown of NPM reversed the drug resistance by down-regulating P-gp and Akt/mTOR signal pathway, indicating that NPM may serve as a potential modulator in drug resistance.