Mucus penetration and intestinal cells targeting are two main strategies to improve insulin oral delivery efficiency. However, few studies are available regarding the effectiveness of combining these two strategies into one nano-delivery system. For this objective, the folic acid (FA) decorated virus-mimicking nanoparticles were designed and influence of FA graft ratio on the in vitro and in vivo properties of insulin loaded nanoparticles was studied systemically. Firstly, using folic acid as active ligand, different folic acid grafted chitosan copolymers (FA-CS) were synthesized and characterized. Thereafter, using insulin-loaded poly(n-butylcyanoacrylate) nanoparticles as the core, virus-mimicking nanoparticles were fabricated by coating of positively charged FA-CS copolymer and negatively charged hyaluronic acid. Irrespective of the FA graft ratio, all the nanoparticles showed good stability, similar insulin release in the gastrointestinal fluid, excellent and similar penetration in mucus. The nanoparticles permeability in intestine was FA graft ratio and segment dependent, with FA graft ratio at/over 12.51% presenting better effect in the order of duodenum > jejunum ≈ ileum. Both mechanism studies and confocal microscopy observation demonstrated FA-mediated process was involved in the transport of FA decorated nanoparticles. In vivo studies revealed hypoglycemic effect of the nanoparticles was FA graft ratio dependent, a saturation phenomenon was observed when FA graft ratio was at/over 12.51%. In conclusion, folic acid decorated virus-mimicking nanoparticles presented improved insulin absorption, implying combining mucus penetration and active transcellular transport is an effective way to promote oral insulin absorption, while the modification ratio of active ligand needs optimization.