The inositol 1,4,5-trisphosphate (InsP3) receptor type I (InsP3R-I) is the principle channel for intracellular calcium (Ca2+) release in many cell types, including central neurons. It is regulated by endogenous compounds like Ca2+ and ATP, by protein partners, and by posttranslational modification. We report that the InsP3R-I is modified by O -linked glycosylation of serine or threonine residues with β- N -acetylglucosamine (O-GlcNAc). The level of O -GlcNAcylation can be altered in vitro by the addition of the enzymes which add [OGT ( O -GlcNActransferase)] or remove ( O -GlcNAcase) this sugar or by loading cells with UDP-GlcNAc. We monitored the effects of this modification on InsP3R function at the single-channel level and on intracellular Ca2+ transients. Single-channel activity was monitored with InsP3R incorporated into bilayers; Ca2+ signaling was monitored using cells loaded with a Ca2+-sensitive fluorophore. We found that channel activity was decreased by the addition of O -GlcNAc and that this decrease was reversed by removal of the sugar. Similarly, cells loaded with UDP-GlcNAc had an attenuated response to uncaging of InsP3. These results show that O -GlcNAcylation is an important regulator of the InsP3R-I and suggest a mechanism for neuronal dysfunction under conditions in which O -GlcNAc is high, such as diabetes or physiological stress.