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Bridging the Atlantic and Mediterranean continental margins, the South Iberian region has recently been the focus for geological and biological investigations. In this region, the Gulf of Cadiz (GoC) encompasses a great variety of deep-sea habitats that harbour highly diverse biological communities. In this study, we describe the composition of gastropod assemblages obtained from in situ colonization experiments and benthic sampling of deep-sea habitats in the GoC. Gastropod distributional patterns, such as bathymetric ranges, bathymetric turnover, affinity to substrate types and abundance-occupancy relationships, are analysed and interpreted in relation to their inferred dispersal capabilities and substrate availability. Overall, the GoC comprises a high diversity of gastropods (65 species), and distinct assemblages were found in typical sedimentary environments at mud volcanoes and in association with carbonate and coral samples or organic substrata. The number of taxa peaked at the Carbonate Province in the middle slope (600–1200 m depth), a highly heterogeneous area with numerous mud volcanoes, carbonate mounds and corals. Darwin (1100 m) and Captain Arutyunov (1300 m) mud volcanoes harboured the most species-rich and abundant gastropod assemblages, respectively. Colonization experiments with organic substrata (wood and alfalfa grass) also yielded diverse and abundant gastropod assemblages. These organic inputs allowed the recruitment of local species but mainly of wood specialist taxa that were not previously known from the GoC. Our results suggest that the distribution of gastropod assemblages may be primarily determined by the occurrence of suitable habitats, probably due to the effect of the substrate type on the structural complexity of the habitat and availability and diversity of adequate food sources. The type of larval development is apparently not a limiting factor for colonization of deep-sea habitats. However, the predominance of non-planktotrophy, and especially lecithotrophy, suggests that a trade-off between more limited dispersal capability and higher potential for self-recruitment may be a recurrent pattern in gastropod species inhabiting reducing environments and other patchily distributed deep-sea habitats. A network of suitable habitats that ensures effective population connectivity would explain the predominance and relatively wide distribution of short-distance dispersing non-planktotrophic species in the GoC deep-sea habitats and other geographical regions.