Enterocytozoon hepatopenaei (EHP) is an important factor limiting the growth of Penaeus vannamei. Its simple transmission route and lack of effective control drugs often lead to great losses for the shrimp aquaculture industry. In this study, some basic information of EHP was provided, including EHP spore purification, germination promoting treatment, quantification determination of the key genes expression, and the activities determination of key enzymes in glycolysis pathway at the stage of spore germination. The result showed that light microscopic and scanning electron microscopic analyses confirmed KOH promoted germination. The expression of the glyceraldehyde 3-phosphate dehydrogenase gene was stable, with high-level expression in treatment groups. Hexokinase gene expression and enzyme activity increased after promoting germination treatment. These findings indicated that the glycolytic pathway is enhanced during EHP germination. The results suggest that we can prevent EHP from invading host cells by attempting to block the energy supply.