This study aimed to analyze the effect of active site mutations on the specificity of bile salt hydrolase (BSH) g1294 from Ligilactobacillus cholophilus BD7642. Glycine scanning technology was used to investigate systematic mutations of BSH g1294’s active center, and the activity of mutants was assessed by the spot-plate method. High performance liquid chromatography was utilized to analyze the specificity of the mutants toward six bile salts. Additionally, saturation mutagenesis was conducted at the N81 site, followed by molecular docking simulations to analyze the differences between the mutants and the wild type. Results revealed that mutations at some sites led to the inactivation of the BSH activity. However, the N81G and N174G mutants exhibited enhanced substrate specificity, the former being superior to the latter. Among the mutants at the N81 site, significant variations in the enzymatic activity and hydrolytic capability were observed. Molecular docking simulations indicated that the N81G mutant acquired hydrolytic capability by reducing the steric hindrance between the catalytic site Cys2 and glycocholic acid. In conclusion, this mutant showed improved specificity than did the wild type. The molecular recognition of bile acids by BSH may not be simply based on amino acid recognition, which could provide new insights for rational design for BSH engineering.