Altered paracrine signaling from the perivascular adipose tissue (PVAT) to the underlying microvasculature is a major determinant of endothelial dysfunction in metabolic syndrome. The composition of PVAT is complex, comprised of both adipocytes and cells of the immune system, however, the relative contribution of individual cell types to vascular dysfunction remains obscure. This study tested the hypothesis that proinflammatory macrophages impinge on microvascular function by reducing the bioavailability of hydrogen sulfide (H2S) in a mouse diet-induced obesity model of metabolic syndrome. Mesenteric resistance arterioles isolated from 20 week-old lean and obese mice were loaded with the fluorescent H2S indicator SF7-AM (5 μM), mounted in a pressure myography chamber, pressurized and imaged confocally to assess [H2S] in the smooth muscle and endothelial layers, and reported as mean ± S.E.M. of background corrected fluorescence units (F). In the smooth muscle layer, [H2S] was 6.4 ± 0.5 F and 3.0 ± 0.2 F in lean and obese respectively. Similarly, [H2S] was lower In the endothelium of vessels from the obese (3.4 ± 0.4 F) compared with lean (6.4 ± 0.4 F), P<0.001, one-way ANOVA, n=8. The same vessels were then precontracted with phenylephrine (1 µM), the vasodilatory response to acetylcholine (0.01-10 µM) measured, and differences between dose-response analyzed as an index of endothelial function. Vasodilation was impaired in vessels from obese animals (P<0.001; two-way ANOVA, n=4). Importantly, incubation with the H2S donor GYY4137 (50 µM) restored H2S levels and endothelial function in obese mouse vessels to levels comparable to those seen in lean controls. To assess the role of macrophages in driving these phenotypes, vessels from lean and obese mice were co-cultured overnight in the presence of macrophages purified by immunomagnetic separation from either lean or obese mice. In vessels from lean mice, [H2S] in both smooth muscle and endothelium was decreased by exposure to macrophages from obese but not lean mice (P<0.001, one-way ANOVA, n=8), which was observed in parallel with impaired vasodilation (P<0.001; two-way ANOVA, n=4). Collectively, these data support a model in which proinflammatory macrophages resident in the PVAT interact with the microvasculature to promote endothelial dysfunction by reducing the bioavailability of H2S.