In this work, the denitrification performance of a bio-ceramsite reactor based on autohydrogenotrophic denitrification was investigated. The effects of various experimental parameters such as nitrate loading, carbon to nitrogen ratio (C/N), water temperature and pH were evaluated during the operation. The unique aspect of this research is that the bio-reactor uses ceramsite as a carrier, which can provide a habitat for autohydrogenotrophic biocoenoses to accrete and grow. The results indicated that the denitrification rate increased as nitrate loading (below 130 mg NO3−-N/L) increased. However, the activity of autohydrogenotrophic denitrifying bacteria was inhibited when nitrate loading was further increased to higher than 130 mg NO3−-N/L. Denitrification efficiency changed slightly with C/N, this system performed well if C/N was more than 0.9. The optimum temperature for the reactor was 25–35 °C. This denitrification system was positively related to pH, as a neutral or alkaline environment was more preferable for the reactor. During the operation, effluent nitrite levels were always maintained below 1.75 mg NO2−-N/L.