Echovirus B serves as one of the important causative agents of viral meningitis and encephalitis all over the world, but no specific vaccines against this virus family are thus far available in the market. Structurally similar to the natural virus whilst lacking the nucleic acid essential for viral replication and infectivity, the virus-like particle (VLP), has widely been considered an ideal vaccine candidate of next generation, especially for the enterovirus that has no envelope. In this study, we designed a platform based on the insect expression system which succeeded in producing the VLP of E16. Then, the 2.8-? cryo-EM structure of E16-VLP was determined and characterized. In line with the overall structure of E30, another member of Enterovirus B, E16-VLP employed the well-studied structural features found in the outer surface of most Enterovirus members and have been regarded to be the regions able to induce specific neutralizing antibodies, indicative of its potential value of vaccine development. Altogether, our established platform could serve as an important candidate to produce a safe and universal vaccine against Enterovirus B.