Aspergillus niger is widely used for enzyme production and achievement of high enzyme production depends on the comprehensive understanding of cells metabolic regulation mechanisms.In this paper, we investigate metabolic differences and regulation mechanisms between a high glucoamylase-producing strain A.niger DS03043 and its wild-type parent strain A.niger CBS513.88 using an integrated isotope-assisted metabolomics and 13C metabolic flux analysis approach.We found that A.niger DS03043 had higher cell growth, glucose uptake, and glucoamylase production rates but lower oxalic acid and citric acid secretion rates.In response to above phenotype changes, A.niger DS03043 was characterized by an increased carbon flux directed to the oxidative pentose phosphate pathway in contrast to reduced flux through TCA cycle, which were confirmed by consistent changes in pool sizes of metabolites.A higher ratio of ATP over AMP in the high producing strain might contribute to the increase in the PP pathway flux as glucosephosphate isomerase was inhibited at higher ATP concentrations.A.niger CBS513.88, however, was in a higher redox state due to the imbalance of NADH regeneration and consumption, resulting in the secretion of oxalic acid and citric acid, as well as the intracellular accumulation of OAA and PEP,which may in turn result in the decrease in the glucose uptake rate.The application of integrated metabolomics and 13C metabolic flux analysis highlights regulation of the energy and redox metabolism on flux redistribution in A.niger.