Sarcoplasmic reticulum (SR) and mitochondria are important intracellular regulators for cytosolic [Ca²⁺], similar to Ca²⁺ channels and pumps at the plasma membrane. However, the functional coupling between SR and mitochondria for regulating cytosolic [Ca²⁺] remains unclear in vascular smooth muscle cells (SMCs). Mitofusins (Mfn1 and Mfn2) are known to tether SR and mitochondria and regulate cytosolic [Ca²⁺]. Mfn1 is expressed in mitochondria membrane, whereas Mfn2 is localized in mitochondria and SR membranes. In the present study, the involvement of mitofusins on functional interaction between SR and mitochondria was examined in rat thoracic aorta SMCs. When the distance between SR and mitochondria was measured using a transmission electron microscope, it was extended from approximately 17 to 24 nm by the siRNA knockdown of Mfn2. Mitochondrial [Ca²⁺] increase by vasopressin (AVP) stimulation was smaller in Mfn2 siRNA-treated SMCs than control cells. On the other hand, SR [Ca²⁺] change was similar in both cells. Ca²⁺ exclusion and uptake after cytosolic [Ca²⁺] increase were delayed in Mfn2 siRNA-treated SMCs. Interestedly, mitochondrial ATP level reduced by the siRNA knockdown of Mfn2. Taken together, Mfn2 facilitates the functional interaction between SR and mitochondria for the regulation of cytosolic [Ca²⁺] in vascular SMCs.