The tick-borne encephalitis (TBE) virus has two membrane glycoproteins (prM and E), which each has one N-linked glycan. Constructs that express prM and E proteins of TBE virus have been shown to produce virus-like particles (VLPs), which have surface properties that are similar to those of infectious viruses. To reveal the function of glycosylation of the TBE virus prM and E proteins in the secretion of VLPs, we expressed glycosylation-mutated prM and E proteins and compared the secretion levels and biological properties of the VLPs. In the prM protein glycosylation-deficient mutant, the level of secreted E protein was reduced to 60% of the wild-type level. On the other hand, in the E or prM-E protein glycosylation-deficient mutant, the level of secreted E protein was reduced to 10% of the wild-type level. Furthermore, the mutant which was glycosylated at positions 66 and 154 in protein E, the level of secreted E protein was four-fold higher than that of the wild-type. However, in the mutant which was glycosylated at position 66 only, E protein secretion was reduced to only 10% of the wild-type level. These data suggest that the glycan associated with the N-linked glycosylation site at position 154 in protein E plays an important role in VLP secretion.