The effects of the potassium (K⁺) channel opener pinacidil (Pin) on the coronary smooth muscle Ca²⁺-myosin light chain (MLC) phosphorylation pathway under hypothermic K⁺ cardioplegia were determined by use of an in vitro microvessel model. Rat coronary arterioles (100-260 µm in diameter) were subjected to 60 min of simulated hypothermic (20°C) K⁺ cardioplegic solutions (K⁺ = 25 mM). We first characterized the time course of changes in intracellular Ca²⁺ concentration, MLC phosphorylation, and diameter and observed that the K⁺ cardioplegia-related vasoconstriction was associated with an activation of the Ca²⁺-MLC phosphorylation pathway. Supplementation with Pin effectively suppressed the Ca²⁺ accumulation and MLC phosphorylation in a dose-dependent manner and subsequently maintained a small decrease in vasomotor tone. The ATP-sensitive K⁺ (K_ATP)-channel blocker glibenclamide, but not the nitric oxide (NO) synthase inhibitor N-nitro-L-arginine methyl ester, significantly inhibited the effect of Pin. K⁺ cardioplegia augments the coronary Ca²⁺-MLC pathway and results in vasoconstriction. Pin effectively prevents the activation of this pathway and maintains adequate vasorelaxation during K⁺ cardioplegia through a K_ATP-channel mechanism not coupled with the endothelium-derived NO signaling cascade.