Two new series of 3-demethoxycarbonyl-3-ester and 3-demethoxycarbonyl-3-ether methyl anhydrovinblastine derivatives were designed, synthesized, and evaluated for their inhibitory activities against human non-small-cell lung cancer (A549) and cervical epithelial adenocarcinoma (HeLa) cell lines. Ester anhydrovinblastine derivatives exhibited potent cytotoxicity, whereas the ether analogues were much less active. The size of the introduced substituents was the foremost factor in determining the resultant cytotoxic activity. Compound 12b showed a similar cytotoxic potency to the positive control, anhydrovinblastine (1a).