The downward flux of particulate organic carbon (POC) decreases significantly in the oceanÂs mesopelagic or ‘twilight’ zone due both to abiotic processes and metabolism by resident biota. Bacteria and zooplankton solubilize and consume POC to support their metabolism, but the relative importance of bacteria vs. zooplankton in the consumption of sinking particles in the twilight zone is unknown. We compared losses of sinking POC, using differences in export flux measured by neutrally buoyant sediment traps at a range of depths, with bacteria and zooplankton metabolic requirements at the Hawaii Ocean Time‐series station ALOHA in the subtropical Pacific and the Japanese times‐series site K2 in the subarctic Pacific. Integrated (150‐1,000 m) mesopelagic bacterial C demand exceeded that of zooplankton by up to 3‐fold at ALOHA, while bacteria and zooplankton required relatively equal amounts of POC at K2. However, sinking POC flux was inadequate to meet metabolic demands at either site. Mesopelagic bacterial C demand was 3‐ to 4‐fold (ALOHA), and 10‐fold (K2) greater than the loss of sinking POC flux, while zooplankton C demand was 1‐ to 2‐fold (ALOHA), and 3‐ to 9‐fold (K2) greater (using our ‘middle’ estimate conversion factors to calculate C demand). Assuming the particle flux estimates are accurate, we posit that this additional C demand must be met by diel vertical migration of zooplankton feeding at the surface and by carnivory at depth—with both processes ultimately supplying organic C to mesopelagic bacteria. These pathways need to be incorporated into biogeochemical models that predict global C sequestration in the deep sea.