To further investigate the potential functions of Streptomyces in the gut, the bile acid-induction experiment was conducted utilizing mass spectrometry for identification. Bile acids (BAs), crucial primary metabolites of cholesterol metabolism, play a vital role in lipid digestion and absorption, enterohepatic circulation, and metabolic regulation. Notably, elevated intestinal bile acid levels are observed during high-fat diet consumption. In the present study, Streptomyces microflavus was stimulated with four distinct bile acids: CA, DCA, CDCA, and UDCA. Subsequently, bacterial cells were harvested, proteins extracted, and data-independent acquisition (DIA)-based proteomic identification performed.