Proteinase-Activated-Receptors (PARs) are a novel family of G-protein coupled receptors (GPCRs) that mediate many biological effects of proteinases. Thrombin activates PAR1, PAR3 and PAR4, whilst trypsin and mast cell tryptase activate PAR2. We have previously reported that N-linked glycosylation provides a subtle but effective mechanism for regulating PAR2 activation. Since PAR1 contains three putative N-linked glycosylation sites on the N-terminus and two on extracellular loop-2, we hypothesised that glycosylation may regulate both proteinase activation and disarming of PAR1. Wild-type hPAR1 and hPAR1eYFP (wt-hPAR1 and wt-hPAR1eYFP) and the glycosylation-deficient mutant receptors (hPAR1N35Q, hPAR1N62Q, hPAR1N75Q, hPAR1N62QN75Q, hPAR1N250Q, hPAR1N259Q, hPAR1N250QN259Q, hPAR1N35-N259Q) were permanently expressed in the Kirsten virus sarcoma transformed rat kidney epithelial (KNRK) cell line. Western blot analysis revealed PAR1 to be an N-linked glycosylated receptor. Flow cytometry and confocal microscopy analysis revealed that removal of only one glycosylation sequon decreases hPAR1 cell surface expression. Removal of all glycosylation sequons resulted in near total retention of the receptor within the cytosol. In calcium signalling assays thrombin and trypsin but not the PAR1-AP, TFLLR-NH2 displayed a reduced ability in activating hPAR1N62QN75Q compared to wt-hPAR1. Further, hPAR1N62QN75Q was strikingly more susceptible to disarming by the neutrophil proteinases proteinase-3, elastase and the bacterial proteinase thermolysin when compared to wt-hPAR1. Thus N-linked glycosylation regulates cell surface expression and proteinase activation and disarming of hPAR1.