Triosephosphate isomerase (TPI) is a vital enzyme in the glycolytic pathway, which can catalyze the interconversion of glyceraldehyde-3-phosphate (GAP) and dihydroxyacetone phosphate (DHAP). DHAP is involved in lipid metabolism and phospholipid synthesis. In order to know the role of TPI in WSSV infection to prawn, we cloned the full length cDNA of triosephosphate isomerase gene (EcTPI) from Exopalaemon carinicauda, and its function during WSSV infection was analyzed. EcTPI transcripts were widely distributed in all tissues, but showed relatively higher expression levels in the gill and epidermis. Its expression was apparently up-regulated after 24 h post WSSV injection (hpi), when the virus load began to rise. Furthermore, we detected the expressions of the key genes encoding the enzymes which catalyze the key steps in the glycolysis during WSSV infection. The data showed that genes encoding the enzymes which catalyzed upper steps of glycolysis to produce GAP, including hexokinase (HK), glucose-6-phosphate isomerase (GPI) and phosphofructokinase-1 (PFK-1), were significantly up-regulated at 24 and 27 hpi. Genes encoding the enzymes catalyzing down steps of glycolysis after GAP, including glyceraldehyde-3-phosphate dehydrogenase (GAPDH), enolase (ENO) and pyruvate kinase (PK), were apparent down-regulated at 24 and 27 hpi. Meanwhile, the gene encoding the enzyme glycerol-3-phosphate dehydrogenase (GPDH) catalyzing DHAP to glycerol-3-phosphate (G-3-P) showed down-regulation at 12–27 hpi, while the gene encoding dihydroxyacetone-phosphate acyltransferase (DHAPAT) catalyzing DHAP to further synthesis of phospholipids showed up-regulation at 12–24 hpi. These data suggested that WSSV infection could change the glycolysis pathway to make them produce more phospholipids which could be very helpful for virus replication. In order to further confirm the above speculation, dsRNA interference (RNAi) approach was used to knock down EcTPI gene and analyze its effect on WSSV load in prawn. The data showed that interference of EcTPI gene led to a significant decrease of WSSV loads in WSSV infected prawn. These data provided useful information to understand the infection mechanism of WSSV.