Calmodulin (CaM) and Ca²⁺/CaM-dependent protein kinase II (CaMKII) play important roles in the development of heart failure. In this study, we evaluated the effects of CaM on mitochondrial membrane potential (ΔΨ_m), permeability transition pore (mPTP) and the production of reactive oxygen species (ROS) in permeabilized myocytes; our findings are as follows. (1) CaM depolarized ΔΨ_m dose-dependently, but this was prevented by an inhibitor of CaM (W-7) or CaMKII (autocamtide 2-related inhibitory peptide (AIP)). (2) CaM accelerated calcein leakage from mitochondria, indicating the opening of mPTP, however this was prevented by AIP. (3) Cyclosporin A (an inhibitor of the mPTP) inhibited both CaM-induced ΔΨ_m depolarization and calcein leakage. (4) CaM increased mitochondrial ROS, which was related to ΔΨ_m depolarization and the opening of mPTP. (5) Chelating of cytosolic Ca²⁺ by BAPTA, the depletion of SR Ca²⁺ by thapsigargin (an inhibitor of SERCA) and the inhibition of mitochondrial Ca²⁺ uniporter by Ru360 attenuated the effects of CaM on mitochondrial function. (6) CaM accelerated Ca²⁺ extrusion from mitochondria. We conclude that CaM/CaMKII depolarized ΔΨ_m and opened mPTP by increasing ROS production, and these effects were strictly regulated by the local increase in cytosolic Ca²⁺ concentration, initiated by Ca²⁺ releases from the SR. In addition, CaM was involved in the regulation of mitochondrial Ca²⁺ homeostasis.