Phospholamban (PLB) inhibits the sarcoplasmic reticulum (SR) Ca²⁺-ATPase (SERCA), and this inhibition is relieved by CaM kinase II (calmodulin-dependent protein kinase II) phosphorylation. We previously reported significant differences in contractility, SR Ca²⁺ release, and CaM kinase II activity in gastric fundus smooth muscles as a result of PLB phosphorylation by CaM kinase II. In this study, we used PLB-knockout mice (PLB-KO) to directly examine the effect of PLB absence on contractility, CaM kinase II activity, and intracellular Ca²⁺ waves in gastric antrum smooth muscles. The frequencies and amplitudes of spontaneous phasic contractions were elevated in antrum smooth muscle strips from PLB-KO mice. Bethanecol increased the amplitudes of phasic contractions in antrum smooth muscles from both control and PLB-KO mice. Caffeine decreased and cyclopiazonic acid (CPA) increased the basal tone of antrum smooth muscle strips from PLB-KO mice, but the effects were less pronounced compared to control strips. The CaM kinase II inhibitor KN-93 was less effective at inhibiting caffeine-induced relaxation in antrum smooth muscle strips from PLB-KO mice. CaM kinase II autonomous activity was elevated, and not further increased by caffeine, in antrum smooth muscles from PLB-KO mice. Similarly, the intracellular Ca²⁺ wave frequency was elevated, and not further increased by caffeine, in antrum smooth muscles from PLB-KO mice. These findings suggest that phospholamban is an important modulator of gastric antrum smooth muscle contractility by modulation of SR Ca²⁺ release and CaM kinase II activity.