Calmodulin (CaM) as a ubiquitous Ca²⁺ sensor interacts with multiple key molecules involved in excitation-contraction (EC) coupling. In the present study, we report that adenoviral expression of a mutant CaM lacking all of its four Ca²⁺-binding sites, CaM(1-4), at a level 6.5-fold over endogenous CaM markedly increases the amplitude and abbreviates the decay time of Ca²⁺ transients and contraction in cultured rat ventricular myocytes. To determine the underlying mechanisms, we examined the properties of L-type Ca²⁺ channels, Ca²⁺/CaM-dependent protein kinase II (CaMKII), and phospholamban (PLB) in the sarcoplasmic reticulum (SR). We found that CaM(1-4) expression markedly augmented L-type Ca²⁺ current amplitude and slowed its inactivation. Surprisingly, overexpression of CaM(1-4) increased CaMKII activity and phosphorylation of PLB-Thr-17. Moreover, CaM(1-4) elevated diastolic Ca²⁺ and caffeine-labile Ca²⁺ content of the SR. Inhibition of CaMKII by KN-93 or a myristoylated autocamtide-2 related inhibitory peptide prevented the aforementioned PLB phosphorylation and reversed the positive inotropic and relaxant effects, indicating that CaMKII is essential to CaM(1-4) actions. These results demonstrate that CaM modulates Ca²⁺ influx, SR Ca²⁺ release, and Ca²⁺ recycling during cardiac EC coupling. A novel finding of this study is that expression of a Ca²⁺-insensitive CaM mutant can lead to activation of CaMKII in cardiac myocytes.