Batch experiments using acclimated sludge to sulfur utilizing autotrophic denitrification were performed to determine the applicability of spent sulfidic caustic in autotrophic denitrification as alternative sulfur and alkalinity sources. Fluorescence in situ hybridization (FISH) analysis showed that the microbial community of β-proteobacteria/Eubacteria increased from 45% to 69% during enrichment period and nitrate removal reached up to 84% under this enriched sludge condition. In thiosulfate utilizing autotrophic denitrification, the initial condition at a sulfur/nitrate (S/N) ratio of 1.5 showed higher nitrate removal with 95.9%, and nitrate removal could be expressed by a first-order function of biomass concentration if all parameters such as pH, alkalinity and S/N ratio were in the optimum range. In spent sulfidic caustic utilizing autotrophic denitrification, the sulfate formation ratios to nitrate reduction were lower than those in thiosulfate utilizing autotrophic denitrification with a range of 2.65 to 2.78, and nitrate removal was over 95% at 1.0 and 1.5 S/N ratios. For S/N ratios of 1.0 and 1.5, initial alkalinities were sufficient to maintain optimum pH range of autotrophic denitrification. Furthermore, well enriched seeding sludge showed good activity of autotrophic denitrification at pH over 10. Therefore, spent sulfidic caustic could be effectively applied to autotrophic denitrification as an alternative sulfur source and an alkalinity source.