Abstract: OBJECTIVE Betulinic acid (BA) from the waste of Nelumbinis Rhizoma (NR) was introduce sugar groups at specific positions by biotransformation. The fermentation products were isolated and purified. Their structural characterization and antitumor activities were also investigated， which may improve utilization of NR. METHODS The content levels of BA in different parts of NR were analyzed by the modified HPLC-PDA method. With the transformation ability of strains screened， the transformation products were analyzed using a UPLC-QTOF-MS method， isolated and purified by silica gel column chromatography repeatedly. The structure characteristics were analyzed by NMR and MS. Cell viability was evaluated by the methylthiazolyl tetrazolium (MTT) assay. RESULTS The content of BA(1) in the nodus was measured to be 0.621%， the highest in the parts of NR. The strain， identified as Bacillus amyloliquefaciens FJ18， was used in the biotransformation. A novel glycosylation derivative of betulinic acid (BA) derivative， 3β-hydroxy-lup-20(29)-en-28-oic acid 28-O-(4-oxy-4-oxobutanoic acid-4-O-β-D-glucopyranosyl) ester， together with a known derivative， 28-O-β-D-glucopyranosyl-3β-hydroxy-lup-20(29)-en-28-oate were obtained by bioconversion of BA. Their structures were determined on the basis of spectroscopic data. The biotransformation products showed in vitro cytotoxic activity against A375 (melanoma)， HeLa (cervical carcinoma)， U251 (glioma)， SH-SY5Y (neuroblastoma) and MCF-7 (breast cancer) cell lines. CONCLUSION The biotransformation of betulinic acid to the derivatives featuring D- glucopyranosyl moieties was accomplished. The in vitro cytotoxicity study revealed that the derivatives exhibit potent cytotoxic activity against cancer cell lines.