Cucurbitacin D, a newly isolated triterpenoid cucurbitacin, has been found to possess anticancer effects. The purpose of this study was to elucidate the effects of cucurbitacin D on human endometrial and ovarian cancer cells. Human endometrial and ovarian cancer cells were treated with various concentrations of cucurbitacin D, and its effects on cell growth, the cell cycle, apoptosis, and their related measurements were investigated in vitro. All endometrial and ovarian cancer cell lines were sensitive to the growth-inhibitory effect of cucurbitacin D. Cell cycle analysis indicated that their exposure to cucurbitacin D increased the proportion in the sub-G0/G1 phases and G2/M phases of the cell cycle. Induction of apoptosis was confirmed by annexin V staining of externalized phosphatidylserine and loss of the transmembrane potential of mitochondria. This induction occurred in concert with altered expression of genes related to cell growth, malignant phenotype, and apoptosis. Our results suggest that cucurbitacin D might be a new therapeutic option for the treatment of endometrial and ovarian cancers.