Zero-valent sulfur (ZVS) and thiosulfate are important intermediates in the biogeochemical cycle of sulfur. The former has been proved to be existed in the cold seep and hydrothermal systems. Three thiosulfate oxidation pathways are already identified and here we found a novel one in Erythrobacter, which was the first genus in the family Erythrobacteraceae that could accumulate ZVS during thiosulfate oxidation. Erythrobacter flavus 21-3 was isolated from the sediment of clod seep. Genomic analyses showed the typical genes encoding Sox multienzyme complex were absent in the genome of E. flavus 21-3. Through proteome and genome data, we identified a three gene involved pathway of thiosulfate oxidation, including thiosulfate dehydrogenase (tsdA), thiosulfohydrolase (soxB) and sulfur dioxygenases (sdo). For the first time, genetic operating system was constructed in Erythrobacter, and mutant strains ΔtsdA, ΔsoxB, ΔsdoA, ΔsdoB and ΔsdoAB were constructed. Stoichiometry was calculated and tetrathionate and ZVS were found to be the intermediates in this novel thiosulfate oxidation pathway. The diverse and distribution of these proteins were investigated and this novel thiosulfate oxidation pathway may exist in bacteria formerly ignored the function of ZVS production. The results give a powerful evidence for a new source of biogenetic ZVS in cold seep.