A general review of the sub-seafloor biosphere is presented. This includes an update and assessment of prokaryotic cell distributions within marine sediments, current deepest 1922 m, and the impact of this on global sub-seafloor biomass estimates. These global estimates appear relatively robust to different calculation approaches and our updated estimate is 5.39 × 1029 cells, taking into consideration new data from very low organic matter South Pacific Gyre sediments. This is higher than other recent estimates, which is justified as several sediments, such as gas hydrate deposits and oil reservoirs, can have elevated cell concentrations. The proposed relationship between elevated cell concentrations and Milankovitch Cycles in sequential diatom rich layers at some sites, demonstrates not only a dynamic deep biosphere, but also that the deep biosphere is an integral part of Earth System Processes over geological time scales. Cell depth distributions vary in different oceanographic provinces and this is also reflected in contrasting biodiversity. Despite this there are some clear common, sub-seafloor prokaryotes, for Bacteria these are the phyla Chloroflexi, Gammaproteobacteria, Planctomycetes and the candidate phylum JS1, and for Archaea uncultivated lineages within the phylum Crenarchaeota (Miscellaneous Crenarchaeotal Group and Marine Benthic Group B), Euryarchaeota (SAGMEG, Marine Benthic Group-D/Thermoplasmatales associated groups) and Thaumarchaeota (Marine Group I). In addition, spores, viruses and fungi have been detected, but their importance is not yet clear. Consistent with the direct demonstration of active prokaryotic cells, prokaryotes have been enriched and isolated from deep sediments and these reflect a subset of the total diversity, including spore formers that are rarely detected in DNA analyses. Activities are generally low in deep marine sediments (~ 10,000 times lower than in near-surface sediments), however, depth integrated activity calculations demonstrate that sub-surface sediments can be responsible for the majority of sediment activity (up to 90%), and hence, are biogeochemically important. Unlike near-surface sediments, competitive metabolisms can occur together and metabolism per cell can be 1000 times lower than in culture, and below the lowest known maintenance energies. Consistent with this, cell turnover times approach geological time-scales (100–1000s of years). Prokaryotic necromass may be an important energy and carbon source, but this is largely produced in near-surface sediments as cell numbers rapidly decrease. However, this and deposited organic matter may be activated at depth as temperatures increase. At thermogenic temperatures methane and other hydrocarbons, plus H2, acetate and CO2 may be produced and diffuse upwards to feed the base of the biosphere (e.g. Nankai Trough and Newfoundland Margin). Temperature activation of minerals may also result in oxidation of sulphides and the formation of electron acceptors, plus H2 from low temperature (~ 55 °C) serpentenisation and water radiolysis. New mineral surface formation from fracturing, weathering and subduction etc. can also mechanochemically split water producing both substrates (H2) and oxidants (O2 and H2O2) for prokaryotes. These and other biosphere:geosphere interactions may be important for sustaining a globally significant sub-seafloor biosphere.