BACKGROUND: Nitrogen and sulfur pollutants have become an urgent problem in surface and groundwater. Short-cut denitrification coupled with anaerobic ammonia oxidation (ANAMMOX) is an economical and efficient biological nitrogen removal process. However, single heterotrophic denitrification and autotrophic denitrification face the problems of high treatment cost and high secondary sulfate pollution, respectively. Moreover, in real environments, single nutrient conditions are less common. Therefore, this work aims to study the process of mixotrophic short-cut denitrification coupled with ANAMMOX to achieve simultaneous nitrogen and sulfur removal.RESULTS: Mixotrophic short-cut denitrification was successfully initiated on day 20, while the nitrite accumulation rate (NAR) was 81.47%. The optimal performance of the mixotrophic denitrification system was achieved when the ratio of sulfur to nitrogen to carbon (S/N/C) was 3/4/9, with S 2− -S, NO 3 − -N, and COD removal reaching 100%, 94.48%, and 95.48%, respectively, with a NAR of 94.24% and only 11.73 mg L −1 of SO 4 2− -S production. Modified Boltzmann and Gompertz models were found to be suitable to describe the removal of NO 3 − -N, with both models exhibiting good data fitting (R 2 > 0.99). In addition, no distinctly dominant genus was found in the mixotrophic denitrification systems. Autotrophic, heterotrophic, and facultative denitrifying bacteria (such as Thiobacillus Pseudomonas and Halomonas) were found to coexist and synergistically perform denitrification. CONCLUSION: Mixotrophic short-cut denitrification can achieve the efficient accumulation of NO 2 − -N and reduce the amount of SO 4 2− -S production at the same time. Mixotrophic short-cut denitrification coupled with ANAMMOX can deeply remove sulfide, nitrogen, and organic carbon.