The metabolic cooperation in the ecosystem of Bacillus megaterium and Ketogulonicigenium vulgare was investigated by cultivating them spatially on a soft agar plate. We found that B. megaterium swarmed in a direction along the trace of K. vulgare on the agar plate. Metabolomics based on gas chromatography coupled with time-of-flight mass spectrometry (GC-TOF-MS) was employed to analyze the interaction mechanism between the two microorganisms. We found that the microorganisms interact by exchanging a number of metabolites. Both intracellular metabolism and cell-cell communication via metabolic cooperation were essential in determining the population dynamics of the ecosystem. The contents of amino acids and other nutritional compounds in K. vulgare were rather low in comparison to those in B. megaterium, but the levels of these compounds in the medium surrounding K. vulgare were fairly high, even higher than in fresh medium. Erythrose, erythritol, guanine, and inositol accumulated around B. megaterium were consumed by K. vulgare upon its migration. The oxidization products of K. vulgare, including 2-keto-gulonic acids (2KGA), were sharply increased. Upon coculturing of B. megaterium and K. vulgare, 2,6-dipicolinic acid (the biomarker of sporulation of B. megaterium), was remarkably increased compared with those in the monocultures. Therefore, the interactions between B. megaterium and K. vulgare were a synergistic combination of mutualism and antagonism. This paper is the first to systematically identify a symbiotic interaction mechanism via metabolites in the ecosystem established by two isolated colonies of B. megaterium and K. vulgare.The biosphere is dominated by microorganisms. Microorganisms usually live together with other organisms and form various ecosystems, such as predator-prey, mutualism, and symbiotic interaction (8). An understanding of symbiotic interaction provides fundamental insights into the screening and production of new natural chemical compounds (26). Symbiotic interaction could be achieved by several strategies, among which metabolic cooperation is one of the most common. Metabolic cooperation is usually achieved by diverse means, including transferring intermediate metabolites, removing limiting by-products, and performing different functions to complete the energy cycling (25).To investigate metabolic cooperation in the ecosystem, the isotope tracer technique, chromatographic separation, and high-throughput chemical analysis techniques were also applied (1,5,10,11,14,28,29). For example, the elementalisotope technique has been used to study the nitrogen transfer pathway (11), phosphorus metabolism in legume-Rhizobium tropici symbiosis (1), and Bacillus detoxification of indolebased inhibitors of Bacillus to enhance the growth of Symbiobacterium thermophilum (29). Recently, new techniques have advanced the study of metabolic cooperation at the system level. Metagenomic approaches were applied to study the function and interaction mechanisms of complex ecosystems, such as the human intest...