The cytoskeleton is believed to have an important role in the structural and functional integrity of endothelial cells. The role of the endothelial cytoskeleton, specifically microtubules, in the mediation of flow-induced dilation of arterioles has not yet been studied. Thus the aim of our study was to investigate the role of microtubules in the endothelial mechanotransduction of flow-induced dilation of isolated gracilis arterioles of the rat. The active diameter of arterioles at a constant perfusion pressure (80 mmHg) was ~63 µm, whereas their passive diameter (Ca²⁺-free solution) was ~119 µm. At a constant pressure, increases in flow of the perfusate solution (from 0 to 10 and from 10 to 20 µl/min) elicited increases in diameter up to ~95 µm (~53% increase). Intraluminal administration of nocodazole at concentrations of 5 × 10⁹ and 5 × 10⁸ M had no discernible effects on the structure of endothelial microtubules or on flow-induced dilation, whereas it disassembled microtubules and eliminated flow-induced dilation at a concentration of 5 × 10⁷ M. At this higher concentration, however, the basal diameter and dilations to acetylcholine (10⁸ M), sodium nitroprusside (10⁷ M), arachidonic acid (5 × 10⁶ M), and prostaglandin E₂ (10⁸ M) were unaffected. Colchicine (5 × 10⁷ M) also disassembled microtubules and eliminated flow-induced dilation. We concluded that, in isolated arterioles, the integrity of the endothelial cytoskeleton is essential for the transduction of the shear stress signal that results in the release of endothelial factors evoking dilation.