It is of great economic interest to produce succinate from low-grade carbon sources, which can enhance the competitiveness of the biological route. In this study, succinate producer Escherichia coli CT550/pHL413KF1 was further engineered to efficiently use the mixed sugars from non-food based soybean hydrolysate to produce succinate under anaerobic conditions. Since many common E. coli strains fail to use galactose anaerobically even if they can use it aerobically, the glucose, and galactose related sugar transporters were deactivated individually and evaluated. The PTS system was found to be important for utilization of mixed sugars, and galactose uptake was activated by deactivating ptsG. In the ptsG(-) strain, glucose, and galactose were used simultaneously. Glucose was assimilated mainly through the mannose PTS system while galactose was transferred mainly through GalP in a ptsG(-) strain. A new succinate producing strain, FZ591C which can efficiently produce succinate from the mixed sugars present in soybean hydrolysate was constructed by integration of the high succinate yield producing module and the galactose utilization module into the chromosome of the CT550 ptsG(-) strain. The succinate yield reached 1.64mol/mol hexose consumed (95% of maximum theoretical yield) when a mixed sugars feedstock was used as a carbon source. Based on the three monitored sugars, a nominal succinate yield of 1.95mol/mol was observed as the strain can apparently also use some other minor sugars in the hydrolysate. In this study, we demonstrate that FZ591C can use soybean hydrolysate as an inexpensive carbon source for high yield succinate production under anaerobic conditions, giving it the potential for industrial application.