To study of structural modification in steroidal saponin by biotransformation technology. Structural modification was carried out of glycosyl residues of dichotomin at C-3 position by Pectinex BE XXL (PBX). Cytotoxicity against HL-60 cells of all products was investigated. The PBX could hydrolyze the glycosyl residues of dichotomin one-by-one to produce nine transformed compounds. The transformed product (mixture) and four secondary spirostanosides among them exhibited cytotoxicity. Compounds P5, P6 and P7 with α-1, 2 end-rhamnosyl residues showed a potential cytotoxic effect on HL-60 cells; P8 with α-1, 4 end-rhamnosyl residue had a weak effect on HL-60 cells. Dichotomin, secondary furostanosides and aglycone were not cytotoxic. PBX has various hydrolyzation to the glycosidic bonds of dichotomin. Nine obtained transformed compounds widen steroidal saponin library. Meanwhile, screening activity affords data for further pharmacological study.