With a particle size comparable to that of small bacteria and a 1.2 Mbp double-strand DNA genome that carries more than 1000 open reading frames, the amoeba-infecting Mimivirus, along with other recently identified members of the Mimiviridae family, are the largest and most complex viruses yet identified. The Mimivirus particle includes an internal membrane that underlies an icosahedral capsid. The assembly mechanism of internal membrane during Mimivirus infection remains unclear, as is the case for other viruses containing internal membranes. By using diverse imaging techniques, we showed that membrane biogenesis is an elaborate process that occurs at the periphery of viral factories generated at the host cytoplasm. This multistage process, which includes the formation of open membrane sheets, enables efficient and continuous assembly of multiple Mimivirus progeny. The membrane biogenesis process suggested here provides novel insights into the assembly of internal viral membranes in general.