[Objective] Ergothioneine, a rare natural amino acid, is a powerful antioxidant with important physiological functions in the body. It has been widely used in the fields of food, medicine, and cosmetics. However, extracting ergothioneine from mushrooms and chemical synthesis suffer from low yields and high costs. This study aims to use metabolic engineering approaches to improve the yield of ergothioneine in Actinoplanes sp. HS. [Methods] Firstly, we locked onto the genes potentially involved in ergothioneine synthesis in Actinoplanes sp. HS by bioinformatics analysis. Then, we identified the functions of these genes by heterologous expression in Escherichia coli BL21(DE3). Finally, the identified functional genes were combined and overexpressed in Actinoplanes sp. HS, and the yields of ergothioneine in the mutant strains were measured. By adding different concentrations of precursors to the fermentation medium, we investigated the impact of precursor concentration on the yield of ergothioneine. [Results] The enzymes encoded by BC03-04016, BC03-04015, BC03-04014, and BC03-04013 in Actinoplanes sp. HS could synthesize the ergothioneine precursor hercynine-cysteine-sulphoxide (HER-Cys-Sul). The enzymes encoded by BC03-04046 and BC03-04917 had the function of cleaving carbon-sulfur bonds, which could catalyze the formation of the final product ergothioneine from HER-Cys-Sul. The mutant strains YC313 and YC314 obtained through overexpression of genes in ergothioneine synthesis showed ergothioneine yields of 125 mg/L and 108 mg/L, respectively, which were 2.9 times and 2.5 times that of the wild-type strain Actinoplanes sp. HS. Additional supplementation of 0.35 g/L methionine in the fermentation medium increased the ergothioneine yield of YC313 by 24% compared with that in the original medium, and the addition of 10 g/L soybean meal resulted in a 19% increase in the ergothioneine yield. [Conclusion] In this study, we identified the genes and their functions in ergothioneine synthesis in Actinoplanes sp. HS and obtained two strains with high yields of ergothioneine through metabolic engineering. Furthermore, we investigated the impact of precursor supply on the yield of ergothioneine, which provided strategic support for the production of ergothioneine by Actinoplanes sp.