The apicomplexan parasite Toxoplasma gondii recognizes, binds and penetrates virtually any kind of mammalian cell using a repertoire of proteins released from late secretory organelles and a unique form of gliding motility (also named glideosome) that critically depends on actin filaments and myosin. How T. gondii glycosylated proteins mediate host-parasite interactions remains elusive. To date, only limited evidence is available concerning N-glycosylation in apicomplexans. Here, we report comprehensive proteomic and glycomic analyses showing that several key components required for host cell-T. gondii interactions are N-glycosylated. Detailed structural characterization confirmed that N-glycans from T. gondii total protein extracts consist of oligomannosidic (Man5-9GlcNAc2) and paucimannosidic (Man3-4GlcNAc2) sugars, which are rarely present on mature eukaryotic glycoproteins. In situ fluorescence using Concanavalin A (Con A) and Pisum sativum agglutinin (PSA) predominantly stained the entire parasite body. Visualization of Toxoplasma glycoproteins purified by affinity chromatography followed by detailed proteomic and glycan analyses identified components involved in gliding motility, moving junction and other additional functions implicated in intracellular development. Importantly, tunicamycin-treated parasites are considerably reduced in motility, host cell invasion and growth. Collectively, these results indicate that N-glycosylation probably participates in modifying key proteins that are essential for host cell invasion by T. gondii.