Aim: This study aimed to investigate the effect of norcantharidin (NCTD) on human mesangial cells (HMCs) apoptosis in vitro and further examine its molecular mechanism. Methods: HMCs were divided into 5 groups: control group, 25% fetal bovine serum (FBS)-treated group, and NCTD groups (NCTD [2.5, 5 and 10 µg/mL] + 25% FBS, respectively). Cell proliferation was determined by MTT assay, while apoptosis was evaluated by Hoechest 33258 staining, the level of cytochrome c, immunohistochemistry, and apoptotic-related proteins/gene expression. Results: Cell viability was inhibited in NCTD-treated HMCs in a dose-dependent manner. The number of apoptotic cells and the content of cytochrome c were significantly increased by NCTD treatment but that of mitochondrial membrane was decreased. Moreover, the expression of bcl-2 and caspase-3 was prompted by NCTD, but the expression of bax, MMP-2, and MMP-9 in 25% FBS-treated HMCs was inhibited. In addition, NCTD markedly unregulated the expression of apoptosis-related gene/protein, including p-Erk1/2, phosphorylated-Jun N-terminal kinase (JNK), p-p38, and p53. Conclusion: NCTD enhances 25% FBS-treated HMC apoptosis in vitro, and this effect may be attributed to the modulation of the ERK, JNK, and p38 mitogen-activated protein kinase signaling pathways.