In order to remove nitrogen effectively from the wastewater with a low C/N ratio, the feasibility of using spent mushroom compost (SMC) hydrolysates as carbon sources for denitrification was investigated in a sequencing batch reactor (SBR). With SMCs supplement, the SBR performance was improved obviously within the 180 days of operation. The total nitrogen removal was promoted from 46.9% to 81-89.4%, and no negative impact induced by different SMCs on the SBR system was observed. The abundance of functional genes including amoA, nirS/K, norB and nosZ in the active sludge was quantified by qPCR, and most of them elevated after SMC was fed. 16S rRNA gene high-throughput sequencing showed that the significant change in microbial community not only promoted pollutants removal but also benefited the stability of the reactor. Therefore, SMC could be an extremely promising carbon source used for nitrogen removal due to its cost-effective and efficient characteristics.