The myogenic response is the intrinsic ability of small arteries to contract in response to increased internal pressure. Although microRNAs have been shown to play an important role in vascular smooth muscle function their importance for the regulation of the myogenic response has not been investigated previously. In this study we used smooth muscle-specific and inducible Dicer KO mice, which are deficient of most microRNAs, and found that the myogenic response was ablated in the small mesenteric arteries of these mice. This was associated with an increased PTEN expression, possibly due to deletion of miR-26a. Increased expression of PTEN, a negative regulator of PI3K/Akt pathway may reduce pressure-induced Akt-phosphorylation and pressure induced calcium influx through voltage gated L-type calcium channels. Furthermore, myogenic tone was rescued by the L-type channel agonist BayK 8644 or by transient stimulation with Angiotensin II (Ang II). The effect of Ang II was dependent on AT1-receptor stimulation and activation of the PI3K/Akt pathway. These results suggest a novel mechanism for regulation of myogenic tone in vascular smooth muscle by microRNAs.