Myosin light chain kinase (MLCK) plays a key role in the regulation of actomyosin contraction in a large variety of cells. Two isoforms have been described: a short isoform, widely expressed in smooth muscle cells; and a long isoform (MLCK210), mainly localized in the endothelium. This study investigated the consequences on different cardiovascular parameters of MLCK210 gene deletion using MLCK210 knockout mice and of pharmacological inhibition of the kinase using a specific MLCK inhibitor. Deletion of MLCK210 did not affect systolic blood pressure and heart rate or echocardiographic measurements. Electrocardiographic analysis showed neither atrio- nor intraventricular conduction or repolarization defects. Ex vivo responses of aortic rings to vasoconstrictor and vasodilator agonists were not modified in MLCK210 null mice. However, deletion of MLCK210 attenuated shear stress-induced dilation and produced changes in the balance of endothelial-relaxing factors of small mesenteric arteries (SMA). In particular, a reduced flow-mediated NO-dependent dilation was observed. However, it was partially compensated by enhanced indomethacin-sensitive dilation. No significant changes were detected in the endothelium-derived hyperpolarizing component of the vasodilator response. The above effects of MLCK210 gene deletion were confirmed in SMA from wild-type mice by the use of the MLCK enzymatic inhibitor MMZ-10–057. In summary, deletion of MLCK210 was not associated with abnormalities of main in vivo cardiovascular parameters in mice. This study demonstrates a role for MLCK210 in the regulation of flow-dependent dilation in SMA.