Poorly controlled diabetes in pregnancy, characterized by hypo- and hyperglycemia, is associated with adverse outcomes. We hypothesized that aberrant glucose levels affect vascular function in pregnancy. The effects of glucose concentration on constriction and endothelium-dependent relaxation in uterine arteries of normal C57BL/6 mice were examined. Ex-vivo arteries from 18 pregnant and 14 non-pregnant mice were mounted on a wire myograph, constricted with phenylephrine and relaxed with incremental doses of acetylcholine, in physiological saline solution containing 5 mmol/L glucose. Arteries were then exposed to solutions with 2, 5, 8 or 12 mmol/L glucose for 30 min and constriction/relaxation repeated. On altering glucose concentrations to 2, 8 or 12 mmol/L, maximal constriction was increased in arteries from pregnant but not from non-pregnant mice (paired t-test, p < 0.05). Endothelium-dependent relaxation was enhanced at 12 mmol/L glucose in arteries from pregnant (two-way ANOVA, p < 0.01), but not from non-pregnant mice. Endothelium-dependent relaxation in the uterine artery was pre-dominantly mediated by a non-nitric oxide/non-prostanoid mechanism, with a smaller contribution from nitric oxide, and no prostanoid-mediated relaxation. In summary, acute changes in glucose concentration alter both constriction and endothelium-dependent relaxation in uterine arteries of normal pregnant mice; these effects are unique to pregnancy.