Sulfur-oxidizing autotrophic denitrification (SO-AD) was investigated in a laboratory-scale moving-bed biofilm reactor (MBBR) at a sewage temperature of 22 °C. A synthetic wastewater with nitrate, sulfide and thiosulfate was fed into the MBBR. After 20 days' acclimation, the reduced sulfur compounds were completely oxidized and nitrogen removal efficiency achieved up to 82%. The operation proceeded to examine the denitrification by decreasing hydraulic retention time (HRT) from 12 to 4 h in stages. At steady state, this laboratory-scale SO-AD MBBR achieved the nitrogen removal efficiency of 94% at the volumetric loading rate of 0.18 kg N·(mreactor3·d)−1. The biofilm formation was examined periodically: the attached volatile solids (AVS) gradually increased corresponding to the decrease of HRT and stabilized at about 1,300 mg AVS·Lreactor−1 at steady state. This study demonstrated that without adding external organic carbon, SO-AD can be successfully applied in moving-bed carriers. The application of SO-AD MBBR has shown the potential for sulfur-containing industrial wastewater treatment, brackish wastewater treatment and the upgrading of the activated sludge system. Moreover, the study provides direct design information for the full-scale MBBR application of the sulfur-cycle based SANI process.