ABSTRACT Anaerobic ammonium oxidation with nitrite reduction to dinitrogen (termed anammox) has been reported to be an important process for removing fixed nitrogen (N) in marine ecosystems and in some agricultural and wetland soils. However, its importance in upland forest soils has never been quantified. In this study, we evaluated the occurrence of anammox activity in two temperate forest soils collected from northeastern China. With ¹⁵ N-labeled NO ₃ ⁻ incubation, we found that the combined potential of the N ₂ production rates of anammox and codenitrification ranged from 0.01 ± 0.01 to 1.2 ± 0.18 nmol N per gram of soil per hour, contributing 0.5% to 14.4% of the total N ₂ production along the soil profile. Denitrification was the main pathway of N ₂ production and accounted for 85.6% to 99.5% of the total N ₂ production. Further labeling experiments with ¹⁵ NH ₄ ⁺ and ¹⁵ NO ₂ ⁻ indicated that codenitrification was present in the mixed forest soil. Codenitrification and anammox accounted for 2% to 12% and 1% to 7% of the total N ₂ production, respectively. Two anammox species, “ Candidatus Brocadia fulgida” and “ Candidatus Jettenia asiatica,” were detected in this study but in very low abundance (as indicated by the hzsB gene). Our results demonstrated that the anammox process occurs in forest soils, but the contribution to N ₂ loss might be low in these ecosystems. More research is necessary to determine the activities of different N ₂ releasing pathways in different forest soils. IMPORTANCE In this study, we examined the anammox activity in temperate upland forest soils using the ¹⁵ N isotope technique. We found that the anammox process contributed little to the N ₂ production rate in the studied forest soil. Two anammox organisms, “ Candidatus Brocadia fulgida” and “ Candidatus Jettenia asiatica,” were detected. In addition, we found that codenitrification was another N ₂ production pathway in forest soils. Our results could contribute to the understanding of soil gaseous N losses and microbial controls in forest soils.