Diosgenin (DSG) has attracted attention recently as a potential anticancer therapeutic agent due to its profound antitumor activity. To better utilize DSG as an antitumor compound, two series of DSG-amino acid ester derivatives (3a-3g and 7a-7g) were designed and synthesized, and their cytotoxic activities against six human cancer cell lines (K562, T24, MNK45, HepG2, A549, and MCF-7) were evaluated. The results obtained showed that a majority of derivatives exhibited cytotoxic activities against these six human tumor cells. Structure-activity relationship analysis revealed that the introduction of l-tryptophan to the C-3 position of DSG and the C-26 position of derivative 5 was the preferred option for these compounds to display significant cytotoxic activities. Among them, compound 7g exhibited significant cytotoxicity against the K562 cell line (IC<sub>50</sub> = 4.41 μM) and was 6.8-fold more potent than diosgenin (IC<sub>50</sub> = 30.04 μM). Further cellular mechanism studies in K562 cells elucidated that compound 7g triggered mitochondrial-related apoptosis by increasing the generation of intracellular reactive oxygen species (ROS) and decreasing mitochondrial membrane potential (MMP), which was associated with upregulation of the gene and protein expression levels of Bax, downregulation of the gene and protein expression levels of Bcl-2 and activation of the caspase cascade. The above results suggested that compound 7g might be considered a promising scaffold for further modification of more potent anticancer agents.