Abstract: With a rich variety of chemical energy sources and steep physical and chemical gradients, hydrothermal vent systems offer a range of habitats to support microbial life. Cultivation-dependent and independent studies have led to an emerging view that diverse microorganisms in deep-sea hydrothermal vents live their chemolithoautotrophic, heterotrophic, or mixotrophic life with versatile metabolic strategies. Biogeochemical processes are mediated by microorganisms, and notably, processes involving or coupling the carbon, sulfur, hydrogen, nitrogen, and metal cycles in these unique ecosystems. Here, we review the taxonomic and physiological diversity of microbial prokaryotic life from cosmopolitan to endemic taxa and emphasize their significant roles in the biogeochemical processes in deep-sea hydrothermal vents. According to the physiology of the targeted taxa and their needs inferred from meta-omics data, the media for selective cultivation can be designed with a wide range of physicochemical conditions such as temperature, pH, hydrostatic pressure, electron donors and acceptors, carbon sources, nitrogen sources, and growth factors. The application of novel cultivation techniques with real-time monitoring of microbial diversity and metabolic substrates and products are also recommended.