In this study, stable high-five insect cell line constitutively expressing rotavirus (RV) VP2 was co-transfected with VP6 and VP7-recombinant plasmids. The presence of RV proteins in stably transfected high-five cells was verified by molecular and protein analyses. To yield self-assembled triple-layered RV-like particles (tlRLPs), a stable insect high-five cell line was generated to produce RV VP6 and VP7 besides VP2. Self-assembled tlRLPs were observed by transmission electron microscopy (TEM), and enzyme-linked immunosorbent assay (ELISA) was used to assess their antigenicity in vivo. The results suggest that the stable transfected high-five cells are able to generate tlRLPs with the efficient antigenicity. J. Med. Virol. 87: 102–111, 2015. © 2014 Wiley Periodicals, Inc.