Virus-like particles (VLPs) have been used for numerous therapeutic applications, especially in vaccination, drug delivery and gene therapy. The recombinant adeno-associated virus (rAAV), a leading candidate in gene therapy with approved products, has also been used as a vaccine scaffold, but high production costs using HEK-293 or insect cells limit its application. Here, we establish intracellular production of AAV VLPs in E. coli. VP3 capsid proteins of AAV serotype 5 (AAV5), the most genetically divergent AAV serotype among known 13 human and non-human primate serotypes, were expressed using a pET vector and VLPs were readily purified via an affinity chromatography. The correct formation of AAV5 VLPs was confirmed by ELISA with an anti-AAV5 intact-capsid antibody and imaging by atomic force microscopy. Transduction, as a proof of biological functionality, was confirmed with an internalization assay into human HeLa cells. Furthermore, to assess the effect of the assembly-activating protein (AAP) on capsid assembly and to increase AAV5 VLP production, we co-transformed AAP5 and AAV5 VP3 genes into E. coli cells using two pET vectors harboring different antibiotic resistance genes. The co-expression indeed improved capsid yield. To our knowledge, this is the first report of AAV VLP production inside E. coli cells. Our finding opens an opportunity to explore AAV VLPs in various biomedical applications and paves the way to investigate rAAV production using E. coli, in which AAV VLPs serve as scaffolds or as starting material for encapsidation of molecules of interest such as DNA for the production of rAAV.