Experimental evolution is now frequently applied to many biological systems to achieve desired objectives. To obtain optimized performance for metabolite production, a successful strategy has been recently developed that couples metabolic engineering techniques with laboratory evolution of microorganisms. Previously, we reported the growth characteristics of three lactate-producing, adaptively evolved Escherichia coli mutant strains designed by the OptKnock computational algorithm. Here, we describe the use of (13)C-labeled experiments and mass distribution measurements to study the evolutionary effects on the fluxome of these differently designed strains. Metabolic flux ratios and intracellular flux distributions as well as physiological data were used to elucidate metabolic responses over the course of adaptive evolution and metabolic differences among strains. The study of 3 unevolved and 12 evolved engineered strains as well as a wild-type strain suggests that evolution resulted in remarkable improvements in both substrate utilization rate and the proportion of glycolytic flux to total glucose utilization flux. Among three strain designs, the most significant increases in the fraction of glucose catabolized through glycolysis (>50%) and the glycolytic fluxes (>twofold) were observed in phosphotransacetylase and phosphofructokinase 1 (PFK1) double deletion (pta- pfkA) strains, which were likely attributed to the dramatic evolutionary increase in gene expression and catalytic activity of the minor PFK encoded by pfkB. These fluxomic studies also revealed the important role of acetate synthetic pathway in anaerobic lactate production. Moreover, flux analysis suggested that independent of genetic background, optimal relative flux distributions in cells could be achieved faster than physiological parameters such as nutrient utilization rate.