Metabolic flux analysis via 13C labeling is a high-throughput technology to quantitatively track metabolic pathways and determine overall enzyme functions in cells. Measuring metabolic fluxes allows us to observe the functional output of the combined transcriptome, proteome and metabolome changes and bridges contemporary functional analyses to the cellular phenotype. Two core techniques are necessary for 13C based metabolic flux analysis: 1) precise measurements of the labeling pattern of targeted metabolites (at concentrations as low as 10 nM) and 2) interpretation of large data sets given by mass spectrometry measurements with a computer model to calculate the metabolic fluxes catalyzed by thousands of cellular enzymes. 13C-based metabolic flux analysis has diverse applications for studying environmental microorganisms which are important for bioremediation or bio-fuel production: 1) We investigated the regulation of central metabolism of Shewanella oneidensis MR-1 under various oxygen conditions and proposed pathways important for MR-1 growth under fully anaerobic conditions. Flux analysis also revealed the robustness of MR-1 central metabolism against environmental stresses (salt stress, genetic perturbation and nano-particle stress). 2) We confirmed that Geobacter metallireducens used a complete TCA cycle under Fe3+ reducing condition and discovered an unusual isoleucine biosynthesis pathway in this bacterium. 3) We investigated the annotated genes in Desulfovibrio vulgaris and found R-type citrate synthase. It has been demonstrated that there is a significant value for 13C-based metabolic flux analysis in many fields.