Ergothioneine is a thiohistidine derivative with potential benefits on many aging-related diseases. The central step of aerobic ergothioneine biosynthesis is the oxidative C–S bond formation reaction catalyzed by mononuclear nonheme iron sulfoxide synthases (EgtB and Egt1). Thus far, only the Mycobacterium thermoresistibile EgtB (EgtBMth) crystal structure is available, while the structural information for the more industrially attractive Egt1 enzyme is not. Herein, we reported the crystal structure of the ergothioneine sulfoxide synthase (EgtBCth) from Candidatus Chloracidobacterium thermophilum. EgtBCth has both EgtB- and Egt1-type of activities. Guided by the structural information, we conducted Rosetta Enzyme Design calculations, and we biochemically demonstrated that EgtBCth can be engineered more toward Egt1-type of activity. This study provides information regarding the factors governing the substrate selectivity in Egt1- and EgtB-catalysis and lays the groundwork for future sulfoxide synthase engineering toward the development of an effective ergothioneine process through a synthetic biology approach.