K2P3.1 mediates background potassium currents, and therefore reduced membrane expression of these channels will impair the function of electrically excitable cells. Understanding the mechanisms by which these channels are trafficked to the cell membrane is of vital importance to the understanding of K2P3.1-related channelopathies. Glycosylation is a critical modulator of sodium and potassium channel gating and trafficking. As K2P3.1 is a predicted glycoprotein with one external N-glycosylation site, we therefore investigated whether N-glycosylation has effects on K2P3.1 membrane expression. The N-glycosylation site was deleted using site-specific mutagenesis. Gel shift assays were performed with wild-type K2P3.1 and the N-glycosylation deficient mutant (K2P3.1N53Q) purified from the membrane fraction of COS-7 cells transiently expressing the channels. The molecular weight of K2P3.1N53Q was lighter compared to wild-type K2P3.1 demonstrating that the N-glycosylation site was occupied and that N-glycosylation accounted for approximately 5-10% of the total mass of K2P3.1. When internal GFP- and external HA-tagged-K2P3.1 and -K2P3.1N53Q were transiently expressed in COS-7 cells approximately equal transfection efficiencies was achieved for both constructs (48% and 43%, respectively). However, wild-type K2P3.1 had enhanced membrane expression compared to K2P3.1N53Q, 89% vs. 61% of transfected cells, respectively, as determined by immunofluoresence using an anti-HA antibody. Chronic inhibition of N-glycosylation using tunicamycin also prevented customary membrane expression of K2P3.1 consistent with that seen for K2P3.1N53Q. These data demonstrate that N-glycosylation is an integral component of K2P3.1 and has an important function in determining K2P3.1 channel stability and/or trafficking to the membrane with important physiological implications.