For all viruses, the initial stages of infection predetermine the success of the entire infection process. The crucial initial steps include receptor binding, cell entry, and genome delivery. In our research, we focus on the cell entry and genome delivery of viruses from the family Picornavirales. To infect the host, picornaviruses utilize the endocytic machinery of the cell. Previously, it was demonstrated that virions undergo structural changes triggered by low pH, receptor binding, or a combination of both. These changes have been attributed to genome delivery into the cell cytoplasm. However, the detailed mechanism was not well understood. In our research, using cryo-electron microscopy methods, we demonstrate how picornaviruses invade the cell and release their RNA in vivo.