Pseudomonas fluorescens CHA0 was found to make a stereoisomeric mixture of pyochelin that promoted growth under iron limitation and induced biosynthetic gene expression in CHA0 but was not recognized by P. aeruginosa, while P. aeruginosa pyochelin was functional in its own background but not in P. fluorescens. The structure of the CHA0 siderophore was determined by mass spectrometry, thin-layer chromatography, NMR, polarimetry, and chiral HPLC as enantio-pyochelin, the optical antipode of P. aeruginosa pyochelin. Chemically synthesized enantio-pyochelin was confirmed active in CHA0. The study shows enantio-pyochelin acts as a species-specific siderophore and signaling molecule, with its potential biosynthetic pathway in CHA0 discussed.