The cycling of organic carbon driven by microbes in the ocean’s interior remains enigmatic because microbial organic carbon demand is higher than the supply as the bioavailability of dissolved organic matter in the deep ocean is low. By linking ‘omics’ approaches to in situ prokaryotic activity measurement, we found that while the lifestyle of deep-sea microbes was primarily particle-associated, the high hydrostatic pressure imposed selection pressures on pressure-sensitive microbes, which may lead to a 100-fold decrease in cell activity. The function and origin of the protein fraction of the organic matter, as revealed from the proteomic structure of the marine microbiome (bacteria, archaea, eukaryotes and viruses), exhibited a size-fractionated pattern in the source of the protein pool in the deep ocean where Gammaproteobacteria and zooplankton debris dominated the dissolved and particulate protein pool, respectively, representing a major carbon source to the deep-sea heterotrophic microbiota. Urea released by zooplankton may provide indirect support to nitrifiers for dark inorganic carbon fixation and further increase the carbon bioavailability. Taken together, we identified the metabolic strategy used by deep-sea prokaryotes and potential organic matter source throughout the water column, which highlights the regeneration pathways of organic matter mediated by the deep-sea microbiome.