Perivascular adipose tissue (PVAT), which reduces vascular contractility, is dysfunctional in male offspring of high-fat diet-fed (HFD) rats, partially due to reduced NO bioavailability. OGlcNAcylation of eNOS decreases its activity; thus we investigated the role of O-GlcNAcylation in the prenatal programming of PVAT dysfunction. Female Sprague-Dawley rats were fed either a control (10% fat) or an obesogenic HFD (45% fat) diet for 12 weeks prior to mating, throughout pregnancy and lactation. Offspring were weaned onto the control diet and were killed at 12 and 24 weeks old. Mesenteric arteries from 12- week old (wo) offspring of HFD dams (HFDO), contracted less to U46619, effects mimicked in control arteries by glucosamine. PVAT from 12 and 24 wo control, but not from HFDO, exerted an anti-contractile effect. Glucosamine attenuated the anti-contractile effect of PVAT in vessels from control but not from HFDO. AMPK activation (with A769662) partially restored an anti-contractile effect in glucosamine-treated control and HFDO PVAT. Glucosamine decreased AMPK activity and expression in HFDO PVAT, although phosphorylated eNOS expression was only reduced in that from males. The loss of anti-contractile effect of HFDO PVAT is likely to result from increased OGlcNAcylation, which decreased AMPK activity and, in males, decreased NO bioavailability.