Subplasmalemmal ion fluxes have global effects on Ca²⁺ signaling in vascular smooth muscle. Measuring cytoplasmic and mitochondrial [Ca²⁺]and [Na⁺], we previously showed that mitochondria buffer both subplasmalemmal cytosolic [Ca²⁺] and [Na⁺] in vascular smooth muscle cells. We have now directly measured sarcoplasmic reticulum [Ca²⁺] in aortic smooth muscle cells revealing that mitochondrial Na⁺/Ca²⁺ exchanger inhibition with CGP-37157 impairs sarcoplasmic reticulum Ca²⁺ refilling during purinergic stimulation. By overexpressing hFis1 to remove mitochondria from the subplasmalemmal space, we show that the rate and extent of sarcoplasmic reticulum refilling is augmented by a subpopulation of peripheral mitochondria. In ATP-stimulated cells, hFis-1–mediated relocalization of mitochondria impaired the sarcoplasmic reticulum refilling process and reduced mitochondrial [Ca²⁺] elevations, despite increased cytosolic [Ca²⁺] elevations. Reversal of plasmalemmal Na⁺/Ca²⁺ exchange was the primary Ca²⁺ entry mechanism following ATP stimulation, based on the effects of KB-R7943. We propose that subplasmalemmal mitochondria ensure efficient sarcoplasmic reticulum refilling by cooperating with the plasmalemmal Na⁺/Ca²⁺ exchanger to funnel Ca²⁺ into the sarcoplasmic reticulum and minimize cytosolic [Ca²⁺] elevations that might otherwise contribute to hypertensive or proliferative vasculopathies.