Background & Aims-Smooth muscle is essential for maintaining homeostasis for many body functions and provides adaptive responses to stresses imposed by pathological disorders. Identified cell signaling networks have defined many potential mechanisms for initiating smooth muscle contraction with or without myosin regulatory light chain (RLC) phosphorylation by myosin light chain kinase (MLCK). We generate tamoxifen-inducible and smooth muscle-specific MLCK knockout (KO) mice and provide direct loss-of-function evidence that shows the primary importance of MLCK in phasic smooth muscle contractions.