ABSTRACT Anammox is one of the most innovative nitrogen removal technologies, while its functional bacteria-anaerobic ammonia-oxidizing bacteria (AAOB) is sensitive to the impurities in the wastewater. In this study, the long-term effects of sulfide at different concentrations (0, 5, 10, 20, 30, 50, 25 mg L−1) on low substrate Anammox process were studied. The results showed that when the sulfide was 25–30 mg L−1, AAOB was well coupled with sulfide-denitrifying bacteria and the total nitrogen removal efficiency (TNRE) reached a maximum of 91.0%. The hydroxylamine oxidoreductase activity and Heme-c reached 1.678 EU g−1 SS and 0.0023 mmol g−1 SS, respectively, with the hzo and nosZ gene concentrations as 2.52 × 108 and 4.45 × 107 copies mL−1. 50 mg L−1 sulfide inhibited the nitrogen removal by AAOB, resulting in the TNRE decreasing to 81.7%. The experimental results provide a reference for the practical application of Anammox in treating sulfur-containing wastewater.