An easy and efficient strategy to prepare betulinic acid esters with various anhydrides was used by the enzymatic synthesis method. It involves lipase-catalyzed acylation of betulinic acid with anhydrides as acylating agents in organic solvent. Lipase from Candida antarctica immobilized on an acrylic resin (Novozym 435) was employed as a biocatalyst. Several 3-O-acyl-betulinic acid derivatives were successfully obtained by this procedure. The anticancer activity of betulinic acid and its 3-O-acylated derivatives were then evaluated in vitro against human lung carcinoma (A549) and human ovarian (CAOV3) cancer cell lines. 3-O-glutaryl-betulinic acid, 3-O-acetyl-betulinic acid, and 3-O-succinyl-betulinic acid showed IC(50)<10 microg/ml against A549 cancer cell line tested and showed better cytotoxicity than betulinic acid. In an ovarian cancer cell line, all betulinic acid derivatives prepared showed weaker cytotoxicity than betulinic acid.