TLR4 knockout (KO) mice are protected from high-fat diet (HFD)-induced vasodilator action of insulin. C57BL/6 mice or TLR4-KO mice were fed normal chow diet (NCD; 10% calorie from fat) or HFD (60% calorie from fat) for 10 wk. Mesentery arterioles were isolated and subjected to videomicroscopic analysis for vasodilation in response to various stimulators, including insulin (A), acetylcholine (Ach; B), and sodium nitroprusside (SNP; C). Vessels isolated from WT HFD mice (C57BL/6J) were impaired to dilate in response to insulin vs. those isolated from WT NCD mice. By contrast, arterioles isolated from both NCD and HFD TLR4-KO mice similarly dilated in response to insulin vs. those isolated from WT NCD mice. However, there were no differences in responses to Ach or SNP. Data suggest that HFD specifically impairs insulin-stimulated vasodilation through a TLR4-dependent mechanism. ###P < 0.001, #P < 0.05 (WT+NCD vs. WT+HFD), ***P < 0.001 (WT+HFD vs. TLR4KO+HFD).