The unique ability of Anammox bacteria to metabolize short-chain fatty acids have been demonstrated. However, the potential contributions of active Anammox species to carbon utilization in a mixotrophic Anammox-denitrification process are less well understood. In this study, we combined genome-resolved metagenomics and DNA stable isotope probing (DNA-SIP) to characterize an Anammox process fed with acetate under COD/TN ratios of around 0.30–0.40 and low nitrogen-loading rates. A draft genome of “Candidatus Jettenia caeni” and a novel species that was phylogenetically close to “Candidatus Brocadia sinica” were recovered. Essential genes encoding the key enzymes for acetate metabolism and dissimilatory nitrate reduction to ammonium were identified in the two Anammox draft genomes. The DNA-SIP revealed that Ignavibacterium, “Candidatus Jettenia caeni,” Thauera, Denitratisoma, and Calorithrix predominantly contributed to organic carbon utilization in the acetate-fed Anammox process. In particular, the “Candidatus Jettenia caeni” accounted for a higher proportion of ¹³C-DNA communities than “Candidatus Brocadia sinica.” This result well confirmed the theory of maintenance energy between the interspecies competition of the two Anammox species under low nitrogen-loading rates. Our study revealed its potential important role of the Anammox genus “Candidatus Jettenia” in the treatment of wastewater containing low organic matter and ammonia.