Anaerobic ammonium oxidation (anammox) process plays a significant role in the marine nitrogen cycle. However, the quantitative importance of this process in nitrogen removal in wetland systems, particularly in natural freshwater wetlands, is still not determined. In the present study, we provided the evidence of the distribution and activity of anammox bacteria in a natural freshwater wetland, located in southeastern China, by using N-15 stable isotope measurements, quantitative PCR assays and 16S rRNA gene clone library analysis. The potential anammox rates measured in this wetland system ranged between 2.5 and 25.5 nmol N-2 g(-1) soil day(-1), and up to 20 % soil dinitrogen gas production could be attributed to the anammox process. Phylogenetic analysis of 16S rRNA genes showed that anammox bacteria related to Candidatus Brocadia, Candidatus Kuenenia, Candidatus Anammoxoglobus and two novel anammox clusters coexisted in the collected soil cores, with Candidatus Brocadia and Candidatus Kuenenia being the dominant anammox genera. Quantitative PCR of hydrazine synthase genes showed that the abundance of anammox bacteria varied from 2.3 x 10(5) to 2.2 x 10(6) copies g(-1) soil in the examined soil cores. Correlation analyses suggested that the soil ammonium concentration had significant influence on the activity of anammox bacteria. On the basis of N-15 tracing technology, it is estimated that a total loss of 31.1 g N m(-2) per year could be linked the anammox process in the examined wetland.