A recombinant beta-glucosidase from Clavibacter michiganensis specifically hydrolyzed the outer and inner glucose linked to the C-3 position in protopanaxadiol (PPD)-type ginsenosides and the C-6 position in protopanaxatriol (PPT)-type ginsenosides except for the hydrolysis of gypenoside LXXV (GypLXXV). The enzyme converted gypenoside XVII (GypXVII) to GypLXXV by hydrolyzing the inner glucose linked to the C-3 position. The substrate-binding residues obtained from the GypXVII-docked homology models of beta-glucosidase from C. michiganensis were replaced with alanine, and the amino acid residue at position 512 was selected because of the changed regioselectivity of W512A. Site-directed mutagenesis for the amino acid residue at position 512 was performed. W512A and W512K hydrolyzed the inner glucose linked to the C-3 position and the outer glucose linked to the C-20 position of GypXVII to produce GypLXXV and F-2. W512R hydrolyzed only the outer glucose linked to the C-20 position of GypXVII to produce F-2. However, W512E and W512D exhibited no activity for GypXVII. Thus, the amino acid at position 512 is a critical residue to determine the regioselectivity for the hydrolysis of GypXVII. These wild-type and variant enzymes produced diverse ginsenosides, including GypXVII, GypLXXV, F-2, and compound K, from ginsenoside Rb-1. To the best of our knowledge, this is the first report of the alteration of regioselectivity on ginsenoside hydrolysis by protein engineering.