ABSTRACT: The diel vertical migration (DVM) of fish provides an active transport of labile dissolved organic matter (DOM) to the deep ocean, fueling the metabolism of heterotrophic bacteria and archaea. We studied the impact of DVM on the mesopelagic prokaryotic diversity of the Red Sea focusing on the mesopelagic deep scattering layer (DSL) between 450-600 m. Despite the general consensus of homogeneous conditions in the twilight zone, we observed variability in physico-chemical variables and distinct seasonal indicator prokaryotes inhabiting the DSL, representing between 2% (summer) to over 10% (winter) of total sequences. The DSL samples diverged from the surrounding mesopelagic waters in multidimensional scaling analysis and were distributed according to depth (47% of variance explained). We identified the sources of diversity that contribute to the DSL using spring depth profiles. On average, 7% was related to probable sinking from the epipelagic, 34% was common among the other mesopelagic waters and 38% was attributable to the DVM, with 21% of species being unique to the DSL. We conclude that the mesopelagic physico-chemical properties shape a rather uniform prokaryotic community, but that the 200 m wide DSL contributes uniquely and in a high proportion to the diversity of the Red Sea mesopelagic. The raw 16S sequences used in this research article are available at https://www.ebi.ac.uk/ena/browser/view/PRJEB49545 as 67 paired fastq sequences with consecutive accession numbers: ERX7411972 ��� ERX7412038. The 2 files stored in this repository represent: a) the clean 16S sequences count and taxonomic affiliation (SILVA132 Database) and b) the metadata associated to each of the 67 samples (lat, long, temperature, salinity, nutrient concentrations, bacterial abundance, bacterial size, etc)