Protein kinase C (PKC) is known to regulate ryanodine receptor (RyR)–mediated local Ca2+ signaling (Ca2+ spark) in airway and vascular smooth muscle cells (SMCs), but its specific molecular mechanisms and functions still remain elusive. In this study, we reveal that, in airway SMCs, specific PKCε peptide inhibitor and gene deletion significantly increased the frequency of Ca2+ sparks, and decreased the amplitude of Ca2+ sparks in the presence of xestospogin-C to eliminate functional inositol 1,4,5-triphosphate receptors. PKCε activation with phorbol-12-myristate-13-acetate significantly decreased Ca2+ spark frequency and increased Ca2+ spark amplitude. The effect of PKCε inhibition or activation on Ca2+ sparks was completely lost in PKCε−/− cells. PKCε inhibition or PKCε activation was unable to affect Ca2+ sparks in RyR1−/− and RyR1+/− cells. Modification of RyR2 activity by FK506-binding protein 12.6 homozygous or RyR2 heterozygous gene deletion did not prevent the effect of PKCε inhibition or activation. RyR3 homogenous gene deletion did not block the effect of PKCε inhibition and activation, either. PKCε inhibition promotes agonist-induced airway muscle contraction, whereas PKCε activation produces an opposite effect. Taken together, these results indicate that PKCε regulates Ca2+ sparks by specifically interacting with RyR1, which plays an important role in the control of contractile responses in airway SMCs.