Pyruvate is an important organic acid that plays a key role in the central metabolic pathway. Manipulating transporters is an efficient strategy to enhance production of target organic acids and a means to understand the effects of altered intracellular pyruvate content on global metabolic networks. Efforts have been made to manipulate mitochondrial pyruvate carrier (MPC) to transport pyruvate into different subcellular compartments in Candida glabrata to demonstrate the effects of the subcellular distribution of pyruvate on central carbon metabolism. By increasing the mitochondrial pyruvate content through enhancing the rate of pyruvate transport into mitochondria， a high central carbon metabolism rate， specific growth rate and specific pyruvate production rate were obtained. Comparing the intracellular pyruvate content of engineered and control strains showed that higher intracellular pyruvate levels were not conducive to improving pyruvate productivity or central carbon metabolism. Plasma membrane expression of MPCs significantly increased the expression levels of key rate-limiting glycolytic enzymes. Moreover， pyruvate production of CGΔura3-Sp-MPC1， CGΔura3-Sp-MPC2， and CGΔura3-Sp-MPC1-Sp-MPC2 increased 134.4%， 120.3%， and 30.0%， respectively. In conclusion， lower intracellular pyruvate content enhanced central carbon metabolism and provided useful clues for improving the production of other organic acids in microorganisms.