JC virus (JCV) is a human polyomavirus and the causative agent of the fatal demyelinating disease progressive multifocal leukoencephalopathy (PML). JCV infection of host cells is dependent on interactions with cell surface asparagine (N)-linked sialic acids and the serotonin 5-hydroxytryptamine(2A) receptor (5-HT(2A)R). The 5-HT(2A)R contains five potential N-linked glycosylation sites on the extracellular N terminus. Glycosylation of other serotonin receptors is essential for expression, ligand binding, and receptor function. Also, glycosylation of cellular receptors has been reported to be important for JCV infection. Therefore, we hypothesized that the 5-HT(2A)R N-linked glycosylation sites are required for JCV infection. Treatment of 5-HT(2A)R-expressing cells with tunicamycin, an inhibitor of N-linked glycosylation, reduced JCV infection. Individual mutation of each of the five N-linked glycosylation sites did not affect the capacity of 5-HT(2A)R to support JCV infection and did not alter the cell surface expression of the receptor. However, mutation of all five N-linked glycosylation sites simultaneously reduced the capacity of 5-HT(2A)R to support infection and altered the cell surface expression. Similarly, tunicamycin treatment reduced the cell surface expression of 5-HT(2A)R. Mutation of all five N-linked glycosylation sites or tunicamycin treatment of cells expressing wild-type 5-HT(2A)R resulted in an altered electrophoretic mobility profile of the receptor. Treatment of cells with PNGase F, to remove N-linked oligosaccharides from the cell surface, did not affect JCV infection in 5-HT(2A)R-expressing cells. These data affirm the importance of 5-HT(2A)R as a JCV receptor and demonstrate that the sialic acid component of the receptor is not directly linked to 5-HT(2A)R.