[Objective] This study aims to investigate the roles of ferredoxin and cystathionine γ-lyase genes located in cysteine metabolic pathway of Clostridium acetobutylicum.[Methods] ClosTron technology is used to inactivate target genes to obtain mutants. The mutants, cultured in the phosphate-limited medium, are maintained at acidogenesis and solventogenesis phase at controlled pH under continuous fermentation condition. The OD and glucose uptake of wild type and mutants are determined.[Results] The Δfer and ΔmccB mutants are obtained by using ClosTron system. The Δfer mutant cannot use sulfate as the sole sulfur source, but it will recover growth by adding sulfite or cysteine in batch fermentation. Moreover, cysteine is harmful to the growth of C. acetobutylicum and the Δfer mutant as the sole sulfur source, and it mainly influences the growth in solventogenic phase. The ΔmccB mutant also grows poorly during acidogenic and solventogenic phases with methionine or cysteine as the sole sulfur source.[Conclusion] Cysteine is a key sulfur-containing compound in the metabolic pathway of C. acetobutylicum and its metabolism is tightly controlled. Ferredoxin participates in its biosynthesis through reduction reactions of sulfate to sulfite; while the cystathionine γ-lyase participates in another cysteine biosynthesis pathway from methionine to cysteine, which is not essential.