Intermittent hypoxia (IH) leads to vascular dysfunction, and O-linked-beta-N-acetylglucosamine (O-GlcNAc)ylation may regulate vascular reactivity through the modulation of intracellular signaling. The present study hypothesized that O-GlcNAc modifications contributed to the vascular effects of acute IH (AIH) and chronic IH (CIH) through the MAPK and Ca2+/calmodulin-dependent kinase II (CaMKII) pathways. Rat aortic and mesenteric segments were incubated with DMSO, O-GlcNAcase (OGA) or O-GlcNAc transferase (OGT) inhibitor under either normoxic or AIH conditions for 3 h, and arterial function was then assessed. Meanwhile, arteries isolated from control and CIH rats were exposed to 3 h of incubation under normoxic conditions using DMSO, OGA or OGT as an inhibitor, before assessing arterial reactivity. CIH was found to increase the expression of vascular O-GlcNAc protein and OGT, phosphorylate p38 MAPK and ERK1/2, and decrease OGA levels, but it had no effects on phosphorylated CaMKII levels. OGA inhibition increased global O-GlcNAcylation and the phosphorylation of p38 MAPK, ERK1/2 and CaMKII, whereas OGT blockade had the opposite effects. OGA inhibition preserved acetylcholine-induced relaxation in AIH arteries, whereas OGT blockade attenuated the relaxation responses of arteries under normoxic conditions or undergoing AIH treatments. However, the impairment of acetylcholine dilation in CIH mesenteric arteries was improved. CIH artery contraction was increased following angiotensin II (Ang II) exposure. Blockade of p38 MAPK and ERK1/2, but not CaMKII, attenuated Ang II-induced contractile responses in CIH arteries isolated from the non-OGT inhibitor-treated groups. OGT inhibition significantly blocked contractile responses to Ang II and abolished the inhibitory effects of MAPK inhibitors. These findings indicated that O-GlcNAcylation regulates IH-induced vascular dysfunction, at least partly by modulating MAPK, but not CaMKII, signaling pathways.