Based on the fact that Timosaponin A-III, a saponin isolated from the rhizome of Anemarrhena asphodeloides, is a promising bioactive lead compound in the treatment of cancer, structural modification at the C3 and C26 positions of sarsasapogenin has always been the focus of our structure-activity investigations. In this paper, we describe the synthesis of a range of new derivatives 5a-5o and the evaluation of their antitumor activities in a panel of six human cancer cell lines using the MTT assay in vitro. The results obtained showed that compounds 5h, 5i, and 5n exhibited significant cytotoxic activities against the six cell lines, being more potent than their parent compound sarsasapogenin. Furthermore, the p-fluorobenzyloxy series of compounds generally exhibited stronger cytotoxicities against all the tested cancer cells compared with the benzyloxy and p-methoxybenzyloxy series, and the substitution of pyrrolidinyl and piperazinyl groups at the C26 position was the preferred option for these compounds to display antitumor activities. Compound 5n exhibited excellent cytotoxic activity against MCF-7 cell line (IC50 = 2.95 μM), and was 16.7-fold more potent than sarsasapogenin. Further studies of the cellular mechanism of 5n showed that it arrested MCF-7 cells at the G2/M phase and induced apoptosis and necrosis. All these results show that it is important to carry out structural modification of sarsasapogenin to obtain some promising derivatives with marked antitumor activities, and the representative compound 5n is a lead compound for further research.