To optimize ginsenosides hydrolyzing b b-glucosidase production from Aspergillus niger, response surface methodology was carried out in two stages. The Plackett-Burman design was achieved to screen the important variables that influence b b-glucosidase production. Among 10 variables (wheat bran, soybean powder, CaCl 2 , ginsenosides, KH 2 PO 4 , MgSO 4 , polyethylene glycol (PEG), medium volume, inoculum size, and stirring speed), it was found that wheat bran, KH 2 PO 4 , and stirring speed had significant effect on b b-glucosidase activity due to very low p-values (pϽ0.05). Subsequently, wheat bran, KH 2 PO 4 , and stirring speed were further optimized using central composite design. The optimal b b-glucosidase production was predicted to be 4650.14 U/ml with the combination of factors (wheat bran, 34.51 g/l; KH 2 PO 4 , 1.78 g/l; stirring speed, 161.60 rpm/min). Finally, under optimal fermentation conditions, ginsenoside Rb 1 was converted to Rd and F 2 by A. niger within 10 min. Little compound K was detected at 30 min, and finally F 2 was completely transformed to compound K within 8 h. The putative conversion pathway of Rb 1 by A. niger was Rb 1 , Rd, F 2 , and compound K.