NKG2D is an activating receptor expressed on several types of human lymphocytes. NKG2D ligands can be induced upon cell stress and are frequently targeted post-translationally in infected or transformed cells, in order to avoid immune recognition. Virus infection and inflammation alter protein N-glycosylation and we have previously shown that changes in cellular N-glycosylation, is involved in regulation of NKG2D-ligand surface expression. The specific mode of regulation through N-glycosylation is however unknown. Here we investigated whether direct N-glycosylation of the NKG2D-ligand, MICA itself is critical for cell-surface expression and sought to identify the essential residues. We found that a single N-glycosylation site (N8) was important for MICA018 surface expression. The frequently expressed MICA allele 008, with an altered transmembrane and intracellular domain, was not affected by mutation of this N-glycosylation site. Mutational analysis revealed that a single amino acid (T24) in the extracellular domain of MICA018 was essential for the N-glycosylation dependency, while the intracellular domain was not involved. The HHV7 immunoevasin, U21, was found to inhibit MICA018 surface expression by affecting N-glycosylation and the retention was rescued by T24A substitution. Our study reveals N-glycosylation as an allele-specific regulatory mechanism important for regulation of surface expression of MICA018 and we pinpoint the residues essential for this N-glycosylation dependency. In addition we show that this regulatory mechanism of MICA surface expression is likely targeted during different pathological conditions.