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Abstract Urea is hypothesized to be an important source of nitrogen and chemical energy to microorganisms in the deep sea; however, direct evidence for urea use below the epipelagic ocean is lacking. Here, we explore urea utilization from 50 to 4000 meters depth in the northeastern Pacific Ocean using metagenomics, nitrification rates, and single-cell stable-isotope-uptake measurements with nanoscale secondary ion mass spectrometry. We find that on average 25% of deep-sea cells assimilated urea-derived N (60% of detectably active cells), and that cell-specific nitrogen-incorporation rates from urea were higher than that from ammonium. Both urea concentrations and assimilation rates relative to ammonium generally increased below the euphotic zone. We detected ammonia- and urea-based nitrification at all depths at one of two sites analyzed, demonstrating their potential to support chemoautotrophy in the mesopelagic and bathypelagic regions. Using newly generated metagenomes we find that the ureC gene, encoding the catalytic subunit of urease, is found within 39% of deep-sea cells in this region, including the Nitrososphaeria (syn., Thaumarchaeota; likely for nitrification) as well as members of thirteen other phyla such as Proteobacteria, Verrucomicrobia, Plantomycetota, Nitrospinota, and Chloroflexota (likely for assimilation). Analysis of public metagenomes estimated ureC within 10–46% of deep-sea cells around the world, with higher prevalence below the photic zone, suggesting urea is widely available to the deep-sea microbiome globally. Our results demonstrate that urea is a nitrogen source to abundant and diverse microorganisms in the dark ocean, as well as a significant contributor to deep-sea nitrification and therefore fuel for chemoautotrophy.