Sulfate (SO4 (2-)) is an often-utilized and well-understood inorganic sulfur source in microorganism culture. Recently, another inorganic sulfur source, thiosulfate (S2O3 (2-)), was proposed to be more advantageous in microbial growth and biotechnological applications. Although its assimilation pathway is known to depend on O-acetyl-l-serine sulfhydrylase B (CysM in Escherichia coli), its metabolism has not been extensively investigated. Therefore, we aimed to explore another yet-unidentified CysM-independent thiosulfate assimilation pathway in E. coli. Delta cysM cells could accumulate essential l-cysteine from thiosulfate as the sole sulfur source and could grow, albeit slowly, demonstrating that a CysM-independent thiosulfate assimilation pathway is present in E. coli. This pathway is expected to consist of the initial part of the thiosulfate to sulfite (SO3 (2-)) conversion, and the latter part might be shared with the final part of the known sulfate assimilation pathway [sulfite -> sulfide (S2-) -> l-cysteine]. This is because thiosulfate-grown Delta cysM cells could accumulate a level of sulfite and sulfide equivalent to that of wild-type cells. The catalysis of thiosulfate to sulfite is at least partly mediated by thiosulfate sulfurtransferase (GlpE), because its overexpression could enhance cellular thiosulfate sulfurtransferase activity in vitro and complement the slow-growth phenotype of thiosulfate-grown Delta cysM cells in vivo. GlpE is therefore concluded to function in the novel CysM-independent thiosulfate assimilation pathway by catalyzing thiosulfate to sulfite. We applied this insight to l-cysteine overproduction in E. coli and succeeded in enhancing it by GlpE overexpression in media containing glucose or glycerol as the main carbon source, by up to similar to 1.7-fold (1207 mg/l) or similar to 1.5-fold (1529 mg/l), respectively.