Abstract: The aim of this study was to investigate the effect of norcantharidin (NCTD) on the proliferation and apoptosis of triple-negative breast cancer cell line MDA-MB-231.Western blot was used to detect the effect of NCTD on the expression levels of apoptosis-related proteins Bax/Bcl-2, cleaved-PARP/PARP/PARP, cleved-caspase-9, cleaved-caspase-3 and MCL-1 in MDA-MB-231 cells.Also, the expression levels of autophagy-related proteins LC3-II/LC3-I, Parkin and PINK1 in MDA-MB-231 cells were measured by Western blot.Flow cytometry was used to measure the effect of NCTD on the changes of mitochondrial membrane potential and mitochondrial reactive oxygen species (ROS).The effect of NCTD on autophagy flow in cells expressing mCherry-EGFP-LC3 was detected by a confocal microscope.Moreover, the effects of NCTD combined with chloroquine (CQ) or 3-methyladenine (3-MA) on the apoptosis of MDA-MB-231 cells were detected by flow cytometry.The results showed that NCTD significantly increased the expression levels of Bax/Bcl-2, cleaved-PARP/PARP, cleaved-caspase-9, cleasved-caspase-3 and LC3-II/LC3-I proteins, and promoted the mitochondrial translocation of Parkin, and blocked the autophagic flow in MDA-MB-231 cells. Moreover, NCTD combined with CQ accelerated apoptosis, while NCTD combined with 3-MA decreased apoptosis.These results suggest that NCTD can induce autophagy accumulation and lead to apoptosis of MDA-MB-231 cells.