The anticancer activities of ginsenosides are widely reported. The structure-activity relationship of ginsenosides against cancer is not well elucidated because of the unavailability of these compounds. In this work, we developed a transformation method to rapidly produce rare dehydroxylated ginsenosides by acid treatment. The optimized temperature, time course, and concentration of formic acid were 120°C, 4 h and 0.01%, respectively. From 100 mg of Rh1, 8.3 mg of Rk3 and 18.7 mg of Rh4 can be produced by acid transformation. Similarly, from 100 mg of Rg3, 7.4 mg of Rk1 and 15.1 mg of Rg5 can be produced. From 100 mg of Rh2, 8.3 mg of Rk2 and 12.7 mg of Rh3 can be generated. Next, the structure-activity relationships of 23 ginsenosides were investigated by comparing their cytotoxic effects on six human cancer cells, including HCT-116, HepG2, MCF-7, Hela, PANC-1, and A549. The results showed that: (1) the cytotoxic effect of ginsenosides is inversely related to the sugar numbers; (2) sugar linkages rank as C-3 > C-6 > C-20; (3) the protopanaxadiol-type has higher activities; (4) having the double bond at the terminal C20-21 exhibits stronger activity than that at C20-22; and (5) 20(S)-ginsenosides show stronger effects than their 20(R)-stereoisomers.