Mitochondrial Ca²⁺ uptake plays an important role for regulating Ca²⁺ signals in vascular smooth muscle cells (SMCs). However, Ca²⁺ affinity of mitochondrial Ca²⁺ uniporter (MCU) is very low. Mitofusin (Mfn) proteins are known to tether endoplasmic reticulum (ER) and mitochondria. Mfn1 is expressed in mitochondria membrane, whereas Mfn2 is localized in mitochondria and ER membranes. In the present study, we examined the physiological roles of Mfn proteins on Ca²⁺ signals in vascular SMCs. SMCs were enzymatically isolated from rat thoracic aorta. The cell proliferation was reduced by siMfn2, but not by siMfn1. The co-localization of mitochondria with sarcoplasmic reticulum (SR) was decreased by siMfn2, but not by siMfn1. In siMfn2-treated cells stimulated by vasopressin (AVP), the peak amplitude of [Ca²⁺]_mito was attenuated, whereas that of [Ca²⁺]_SR was not changed. The half duration of [Ca²⁺]_cyt increase was increased by siMfn2. These results indicate that Mfn2 regulates mitochondrial Ca²⁺ uptake without changing the parameters of Ca²⁺ release from SR. In conclusion, Mfn2 enhances Ca²⁺ signal through tethering SR to mitochondria, which promotes vascular SMC proliferation.