Inhibitor kappa B kinase 2 (IKK2) plays an essential role in the activation of nuclear factor kappa B (NF-kB). Recently, it has been suggested that IKK2 acts as a myosin light chain kinase (MLCK) and contributes to vasoconstriction in mouse aorta. However, the underlying mechanisms are still unknown. Therefore, we investigated whether IKK2 acts as a MLCK or regulates the activity of myosin light chain phosphatase (MLCP). Pressure myograph was used to measure vascular tone in rat mesenteric arteries. Immunofluorescence staining was performed to identify phosphorylation levels of MLC (ser19), MYPT1 (thr853 and thr696) and CPI-17 (thr38). SC-514 (IKK2 inhibitor, 50 μM) induced relaxation in the mesenteric arteries pre-contracted with 70 mM high K+ solution or U-46619 (thromboxane analog, 5 μM). The relaxation induced by SC-514 was increased in the arteries pre-contracted with U-46619 compared to arteries pre-contracted with 70 mM high K+ solution. U-46619-induced contraction was decreased by treatment of SC-514 in the presence of MLCK inhibitor, ML-7 (10 μM). In the absence of intracellular Ca2+, U-46619 still induced contraction, which was decreased by treatment of SC-514. Furthermore, phosphorylation levels of MLC (ser19) and MYPT1 (thr853) were decreased by treatment of SC-514. IKK2 is involved in the vascular contraction through regulation of MLCP activity by phosphorylating MYPT1 at thr853 in rat mesenteric arteries. These findings suggest IKK2 could be a new pharmacological target for specific therapies of various vascular diseases.