Nitrogen is one of the most important elements involved in ecosystem biogeochemical cycling. However, little is known about the characteristics of nitrogen cycling during the ice-covered period in seasonally frozen lakes. In this study, shotgun metagenomic sequencing of subglacial water and sediment from Lake Ulansuhai was performed to identify and compare nitrogen metabolism pathways and microbes involved in these pathways. In total, ammonia assimilation was the most prominent nitrogen transformation pathway, and Bacteria and Proteobacteria (at the domain and phylum levels, respectively) were the most abundant portion of microorganisms involved in nitrogen metabolism. Gene sequences devoted to nitrogen fixation, nitrification, denitrification, dissimilatory nitrate reduction to ammonium, and ammonia assimilation were significantly higher in sediment than in surface and subsurface water. In addition, 15 biomarkers of nitrogen-converting microorganisms, such as Ciliophora and Synergistetes, showed significant variation between sampling levels. The findings of the present study improve our understanding of the nitrogen cycle in seasonally frozen lakes.