Objective To study the effects and mechanism of norcantharidin (NCTD) on proliferation and apoptosis of human leukemia NB4 and K562 cells by targeting phosphoprotein phosphatase 5 catalytic (PPP5C). Methods NB4 and K562 cells were cultured in vitro, and the pcDNA3.1 and PPP5C-pcDNA3.1 plasmids were electroporated into NB4 and K562 cells. NB4 and K562 stable cell lines were screened with geneticin (G418). The protein and mRNA expression levels of PPP5C were identified by Western blot and qRT-PCR. The proliferation ability, migration ability and apoptosis rate of NB4、K562 cells were determined by CCK-8 assay, transwell assay and the Live DeadTM animal cell viability/toxicity detection kit. NB4 and K562 cells were divided into control group and different doses of NCTD group, and cultured in 1640 medium containing 0, 8, 16, 32 μg/ml NCTD. The Live DeadTM animal cell viability/toxicity detection kit detected the number of dead and live cells and cell morphology was recorded by microscope. Western blot was used to detect the protein expression levels of caspase 3, Cleaved caspase 3, JNK, p-JNK, p38, p-p38 and α-tubulin in cells of each group. Results? the proliferation ability of cells, the migration ability and apoptosis rate of NB4、K562 cells are enhanced after overexpression of PPP5C in human leukemia NB4 and K562 cells; Compared with the control group, each concentration group of NCTD promoted apoptosis in a dose-dependent manner; PPP5C overexpression antagonizes the killing effect of NCTD on leukemia cells. Conclusions NCTD can promote the apoptosis of NB4 and K562 cells and inhibit the proliferation of cells by inhibiting PPP5C.