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To explore the potential metabolites from rabeprazole sulfide, seven strains of filamentous fungi were screened for their biotransformation abilities. Among these strains, <i>Cunninghamella blakesleeana</i> 3.970 exhibited the best result. Four different culture media were screened in order to identify the most optimal for subsequent research. Single factors such as the initial pH of culture media, culture time, inoculation volume, and media volume were individually investigated to provide the optimum biotransformation conditions. Then, an orthogonal optimization process using a five-factor, four-level L₁₆(4⁵) experiment was designed and performed. Finally, when the substrate concentration is 3 g/L, one major metabolite was detected with a transformation rate of 72.4%. Isolated by semipreparative HPLC, this metabolite was further detected by ESI-MS and NMR. The final data analysis indicated that the metabolite is <i>O</i>-demethylation rabeprazole sulfide.