Small resistance size arteries are key players in vascular resistance and overall blood pressure control. We previously demonstrated that within endothelial cells (EC), H2S-mediated vasodilation involves activation of transient receptor potential cation channel subfamily V member 4 (TRPV4)-dependent Ca2+ influx to activate nearby large-conductance Ca2+-activated potassium (BK) channel in resistance-size arteries. Comparison of vasodilatory responses in large and small (resistance-size) arteries show that small arteries dilate at lower concentrations of H2S donors vs large arteries. The importance of endogenous H2S in endothelium-dependent vasodilatory responses in small resistance size arteries highlights a role for H2S in regulating blood pressure and flow. However, little is known of the differences in H2S signaling in large and small arteries and how these differences modulate vasodilation. Based on previous reports that cell membrane cholesterol negatively regulates TRPV4 mobility and BK activity, we hypothesized that H2S-mediated vasodilation is disrupted by elevated EC membrane cholesterol in large arteries. We first verified EC plasma membrane cholesterol content was higher in large vs small arteries by immunofluorescence using the cholesterol stain, filipin III (20 µg/ml) in EC labeled with the glycocalyx marker tomato lectin (20 µg/ml) and the nuclear stain SYTOX green (large 145.20 ± 13.67, small: 59.85 ± 5.50, p = 0.0004, n = 5 animals/group). Next, we examined the effect of cholesterol depletion using methyl β cyclodextrin (MBCD, 100 μM), on dilatory responses to H2S in large (300-380 μm) and small (60-130 μm) mesenteric arteries using the H2S donor, NaHS (1, 10 or 100 μM). In large arteries, MBCD pretreatment significantly enhanced H2S-mediated vasodilation (10 μM +vehicle: 0.72% ± 2.17, +MBCD: 9.71% ± 2.710, p = 0.026), (100 μM +vehicle: 2.25% ± 5.74, +MBCD: 20.55% ± 7.687, p < 0.0001) n = 5 animals/group. Although NaHS dilated small arteries more than large arteries, there was no effect of cholesterol depletion in small arteries (1 μM +vehicle: 16.25% ± 2.36, +MBCD: 16.29% ± 4.44, NS), (10 μM +vehicle: 33.22% ± 7.10, +MBCD: 39.00% ± 5.85, NS), (100 μM +vehicle: 59.84% ±5.98, +MBCD: 63.18% ± 4.639, NS) n = 3 animals/group. Additionally, immunofluorescence studies of EC TRPV4 expression showed no difference between large and small arteries (large 39.6 ± 12.9, small 41.8 ± 14.1, NS) n =3 animals/group. These studies suggest that membrane cholesterol disrupts H2S-mediated vasodilation and contributes to the relative differences in sensitivity to H2S-mediated vasodilation in large and small arteries.