ABSTRACT Anaerobic ammonia oxidation (anammox) process was achieved in a constructed rapid infiltration (CRI) system and the effect of organic matter on the anammox performance and microbial community structure was investigated. The results showed that the removal efficiencies of -N, N and TN were 99.7 ± 0.3%, 99.8 ± 0.2% and 91.3 ± 0.2% respectively after 83 days of acclimation without the presence of organic matter in the influent. The average TN removal efficiency increased by 3.2%–7.7% due to the synergistic effect of anammox and denitrification at a low level of organic matter concentration (10–30 mg COD/L). At medium or high organic matter concentration (50–100 mg COD/L), denitrification gradually replaced anammox as the predominant nitrogen removal route due to its stronger ability to compete with substrate, resulting in a significant decline in anammox activity. The contribution rate of anammox to nitrogen removal dropped by 70.3% with the influent COD increased from 0 to 100 mg/L, and the TN removal efficiency decreased to 68.4 ± 3.6% since the anammox was seriously suppressed. 16S rRNA high-throughput sequencing analysis illustrated that the genus Candidatus Kuenenia was the predominant anammox bacteria (AAOB) with a relative abundance of 12.63% when no organic matter was applied. While the heterotrophic denitrifying bacteria (DNB) Thauera gradually dominated the community with the elevated organic matter introduction. The findings of this study provide useful information for the stable operation and optimal regulation of anammox in the CRI system when the influent contains organic matter.