Influenza virus-like particles (VLPs) represent an attractive alternative to traditional influenza vaccine formulations. Influenza VLPs mimic the natural virus while lacking the genetic material, are easily recognized by the immune system, and are considered safe. The use of a mammalian cell platform offers many advantages for VLP production, such as flexibility and the same glycosylation patterns as a human virus. In this study, the influenza VLPs containing hemagglutinin (HA), neuraminidase (NA) and matrix M1 proteins were expressed in CHO-K1, Vero or 293?T cell lines using transient transfection. After production in 3L bioreactor and purification, extensive characterization was performed on two batches of VLPs produced in 293?T, the best cell line for VLP expression; one batch expressed the HA and NA genes from A/Hong Kong/4801/2014 (H3N2) strain and the other, HA and NA genes from B/Phuket/3073/2013. Characterizations provided evidence that mammalian VLPs closely emulate the exterior of authentic virus particles in terms of both antigen presentation and biological properties. The two VLPs produced contained more NA proteins on their surface with a HA:NA ratio around 1:1 than influenza viruses which present a HA:NA ratio of around 4:1. Immunogenicity studies in BALB/c mice demonstrated that the VLPs, administered intra-muscularly, were highly immunogenic at low doses, with the induction of functional antibodies against HA and NA. Immunogenicity was also shown in a human in vitro model (MIMIC? system). In conclusion, we believe that influenza vaccines made of VLPs produced in mammalian cell lines, constitute a potential alternative to the classical influenza vaccines.