ABSTRACTZero-valent sulfur (ZVS) distributes widely in the deep-sea cold seep, which is an important immediate in the sulfur cycle of cold seep. In our previous work, we described a novel thiosulfate oxidation pathway determined by thiosulfate dehydrogenase (TsdA) and thiosulfohydrolase (SoxB) mediating the conversion of thiosulfate to ZVS in the deep-sea cold seep bacterium Erythrobacter flavus 21-3. However, the occurrence and ecological role of this pathway in the deep-sea cold seep were obscure. Here, we cultured E. flavus 21-3 in the deep-sea cold seep for 10 days and demonstrated its capability of forming ZVS in the in situ field. Based on proteomic, stoichiometric analyses and microscopic observation, we found that this thiosulfate oxidation pathway benefited E. flavus 21-3 to adapt the cold seep conditions. Notably, ~25% metagenomes assembled genomes derived from the shallow sediments of cold seeps contained both tsdA and soxB, where presented abundant sulfur metabolism-related genes and active sulfur cycle. Our results suggested that the thiosulfate oxidation pathway determined by TsdA and SoxB existed across many bacteria inhabiting in the cold seep and frequently used by microbes to take part in the active cold seep biogeochemical sulfur cycle.