Anaerobic ammonia oxidation (anammox) is one of the most innovative discoveries in field of microbial nitrogen cycle with its direct oxidation of ammonia to nitrogen gas with no N₂O production. At present the anammox researches are limited to two areas:biological wastewater treatment and marine ecosystems. Evidence for anammox was obtained from Baiyangdian, the largest freshwater lake in North China, using an isotope pairing technique and molecular analysis. This lake is characterized by hyper-eutrophic conditions with a great amount of accumulated ammonia nitrogen (95-336 mg/kg) in surface sediments (0-5cm). The anammox processes were detected on an extensive spatial scale (n=10) in the lake sediments by PCR and 16S rRNA gene clone libraries, using primers combination pla46f-630r and Amx368f-Amx820r. The 30 sequences sharing 98% similarity were grouped into only five operational taxonomic unit (OTU) using software DOTUR by the furthest neighbor approach. The biodiversity indicator "Shannon (1.4) and Chao 1 estimator(5)" were also calculated with DOTUR software. The result of biodiversity analysis showed that anammox bacteria biodiversity in the lake sediments was low. Phylogenetic trees were constructed by neighbor-joining (NJ) with the Jukes-Cantor correction in MEGA 4 package. The 16S rRNA gene sequences of anammox bacteria in the surface sediments were closely related to the known anammox bacteria Candidatus Brocadia fulgida (97%) and Candidatus Brocadia anammoxidans (96%), and it revealed most of anammox bacteria from Baiyangdian Lake may belong to "Brocadia". Isotope analyses were based on ¹⁵N labeled ammonium and nitrate. Three treatments (1) ¹⁵NH₄⁺ (negative control) (2) ¹⁵NH₄⁺ and ¹⁴NO₃^- (positive control) (3) ¹⁴NH₄⁺ and ¹⁵NO₃^- (test group) was prepared and experiments were operated in hermetic anaerobic containers at 19℃. Outcome gaseous N₂ was delivered to Gas chromatography-isotope ratio mass spectrometry for separation of ²⁸N₂, ²⁹N₂ and ³⁰N₂. Then the rates of anammox and denitrification activities were calculated. The anammox rates ranged between 0.19 to 7.78 nmol N g^-1 h^-1 with significant spatial heterogeneity, which contributed 0.64 to 20.65 precent to sediments N₂ production with the remainder being due to denitrification. It was estimated that the total removed N (1.8-78 gN m^-2 a^-1) was linked to anammox in this eutrophic freshwater lake. About 56 to 9426 t/a of N was removed by way of anammox process with the water level changing from 5.5 to 9.5 m of Baiyangdian Lake. This research provided some new knowledge on anammox in freshwater ecosystems.