Background – Microtubules are key components of the cell cytoskeleton involved in many biological processes, including cell division and intracellular transport. Although little is known about the role of microtubules in smooth muscle, recent work has shown that the microtubule network can regulate arterial tone by controlling Kv7.4 channel membrane expression in smooth muscle cells. However, the precise mechanism underlying microtubule transport of Kv7.4 channels is yet to be determined. Dyneins are small molecular motor proteins that bind to microtubules and walk toward the minus ends. They are essential for the transport and distribution of certain membrane proteins. The role of dynein in arterial smooth muscle cells is unknown; therefore the aim of this study was to investigate whether dynein was involved in the microtubule-dependent regulation of Kv7.4 in vascular smooth muscle. Methods and results – Experiments were performed on 15- to 18-week-old male Wistar rats euthanized by cervical dislocation. Isometric tension recordings were performed on third-order mesenteric artery segments (~2 mm) in a wire myograph. Mesenteric artery segments were incubated with the dynein inhibitor ciliobrevin, after which either the β-adrenoceptor agonist, isoprenaline, or Kv7.2-7.5 activator, S-1, were applied at increasing concentrations to precontracted artery segments. Treatment with ciliobrevin enhanced isoprenaline- and S-1-mediated relaxations compared to non-treated controls. Morpholino-induced knockdown of Kv7.4 channels prevented ciliobrevin from enhancing S-1-mediated relaxations and partially attenuated the enhanced isoprenaline relaxations. Structured Illuminescence Microscopy on myocytes isolated from arteries showed that dynein and Kv7.4 can locate in close proximity to each other. Given that Kv7.4 channel knockdown only partially attenuated ciliobrevin-enhanced isoprenaline-mediated relaxations, we investigated if there was also a vasorelaxant component unrelated to Kv7.4 channels that was enhanced by dynein inhibition. Isoprenaline stimulation in presence of bisoprolol (β1-adrenergic antagonist) inhibited isoprenaline relaxations in control artery segments. However, when artery segments were incubated with ciliobrevin, bisoprolol had no effect on the relaxations. Conclusion – This study is the first to show regulatory effects of dynein on β-adrenoceptor-mediated relaxations of rat mesenteric arteries. We show that dynein inhibition enhanced Kv7.4 channel function, which contributed to increased β-adrenoceptor mediated relaxations. In addition, dynein inhibition may prevent agonist-induced internalisation of the β2-adrenoceptor, thus increasing isoprenaline-mediated relaxations through mechanisms independent of Kv7 channel recruitment.