A lasso peptide biosynthetic gene cluster (BGC) was identified through genome mining in the species Streptacidiphilus jiangxiensis CGMCC 4.1857, which was isolated from acidic rhizosphere soil. The BGC was reconstructed in Escherichia coli, leading to the heterologous production of a lasso peptide named streptacidin. The solution structure of streptacidin was determined based on the distance constraints derived from the NOESY spectrum, establishing the first lasso peptide utilizing methionine as a steric lock for the unique spatial configuration and the remarkable stability against heat (up to 95 °C) and proteolytic degradation by carboxypeptidase Y and chymotrypsin. Under experimental conditions, streptacidin showed weak effectiveness against vancomycin sensitive Enterococcus faecalis ATCC 29212 and Pseudomonas aeruginosa ATCC 902. These findings further the understanding of this distinctive class of compounds.