Six series of novel isosteviol derivatives; modified in the C-19 position; were synthesized; and their antiproliferative activity was evaluated against three human cancer cell lines (HCT-116; BEL-7402; HepG2) and the human L02 normal cell line in vitro. Most of the derivatives tested here exhibited improved antiproliferative activity with high selectivity when compared with the parent compound isosteviol and the positive control drug 5-fluorouracil. Among these derivatives; compound exhibited the most potent antiproliferative activity and commendable selectivity between cancer and normal cells. In addition; compound inhibited the colony formation of HCT-116 cells in a concentration-dependent manner. Further studies revealed that compound arrested the HCT-116 cell cycle in the S phase; and western blot analysis demonstrated the mechanism may be correlated with a change in the expression of cyclin A; cyclin B1; and cyclin E1. Furthermore; the results of a docking study that involved placing compound into the CDK2/cyclin A binding site revealed that its mode of action was possibly as a CDK2/cyclin A inhibitor.