Diosgenin, a naturally occurring steroidal saponin, has been confirmed to possess potent anticancer properties. In the current work, two series of novel diosgenin derivatives bearing 1,3,4-oxadiazole (6a-6e and 7a-7e) or 1,3,4-thiadiazole (8a-8e and 9a-9e) moieties were designed, synthesized and evaluated for their cytotoxicities in four human cancer cell lines (HepG2, A549, MCF-7 and HCT-116) and normal human gastric epithelial cells (GES-1) using the MTT assay in vitro. The results showed that compounds 8d and 9d exhibited significant cytotoxic activities against the HepG2 and A549 cells, being more potent than their parent compound diosgenin. Furthermore, the 1,3,4-thiadiazole series of compounds generally exhibited stronger cytotoxicity compared with the 1,3,4-oxadiazole series against HepG2 and A549 cells, and the substitution of 3-pyridyl group at the C5 position of the 1,3,4-thiadiazole ring was the preferred option for these compounds to display significant cytotoxic activities. Compound 8d showed potent cytotoxic activity against A549 cell line (IC = 3.93 μM) and was 6.7-fold more potent than diosgenin (IC = 26.41 μM). Moreover, compound 8d displayed low toxicity against GES-1 cells (IC = 420.4 μM), showing specificity between normal and tumor cells. Further cellular mechanism studies in A549 cells indicated that compound 8d triggered the mitochondrial-mediated apoptosis by decreasing mitochondrial membrane potential, which was associated with up-regulation of Bax, down-regulation of Bcl-2 and activation levels of the caspase cascade. The above results indicated that compound 8d may be used as a promising skeleton for antitumor agents with improved efficacy.