Bile salt hydrolase (BSH), synthesized by some intestinal bacteria, catalyzes the hydrolysis of taurine and glycine-linked bile salts in human intestine. Because of the link between BSH and human health, the structural and biochemical features of BSHs necessitate an intensive study to better understand their roles in regulating bacterial and host metabolism. Five amino acids, supposed to be responsible for catalytic activity, are strictly conserved in all members of the N-terminal nucleophile (Ntn) hydrolase superfamily. In order to analyze the correlation between two of these strictly conserved amino acids and catalytic activity of BSH, the asparagine-170 (N170) and arginine-223 (R223) amino acids were substituted for the valine-170 (V170) and phenilalanine-223 (F223), respectively by polymerase chain reaction (PCR)-based site-directed mutagenesis. The mutant recombinant BSHs (mrBSHs) were expressed in Escherichia (E.) coli BLR(DE3). While the effect of the mutations on catalytic activity was detected by the ninhydrin assay, the effect of the mutations on formation of the BSHs was observed by sodium dodecyl sulfate poly acrylamide gel electrophoresis (SDS-PAGE) analysis. It was found that V170 and F223 mutations resulted in lack of BSH expression or stability. This is the first experimental report showing that the N170 and R223 may be critical amino acids for folding or stability of the BSH.