<p>The formation of donut-shaped penetration pore upon membrane fusion in a closed lipid membrane system is of biological significance, since such the structures extensively exist in living body with various functions. However, the related formation dynamics is unclear because of the limitation of experimental techniques. This work developed a new model of intra-vesicular fusion to elaborate the formation and stabilization of penetration pores by employing molecular dynamics simulations, based on simplified spherical lipid vesicle system, and investigated the regulation of membrane lipid composition. Results showed that penetration pore could be successfully formed based on the strategy of membrane fusion. The ease of intra-vesicular fusion and penetration pore formation was closely correlated with the lipid curvature properties, where negative spontaneous curvature of lipids seemed to be unfavorable for intra-vesicle fusion. Furthermore, the inner membrane tension around the pore was much larger than other regions, which governed the penetration pore size and stability. This work provided basic understanding for vesicle penetration pore formation and stabilization mechanisms.</p>