Two -isoforms of the Na⁺-K⁺ ATPase are expressed in vascular smooth muscle cells (VSMC). The 1-isoform is proposed to serve a cytosolic housekeeping role while the 2-isoform modulates Ca²⁺ storage via coupling to the Na⁺-Ca²⁺ exchanger (NCX) in a subsarcolemmal compartment. To evaluate the ramifications of this proposed interaction, Ca²⁺ store load and the contributions of the primary Ca²⁺ transporters to Ca²⁺ clearance were studied in aortic VSMC from embryonic wild-type (WT) and Na⁺-K⁺ ATPase 2-isoform gene-ablated, homozygous null (2KO) mice. Ca²⁺ stores were unloaded by inhibiting the sarco-endoplasmic reticulum Ca²⁺-ATPase with cyclopiazonic acid (CPA) in Ca²⁺-free media to limit Ca²⁺ influx. Ca²⁺ clearance by the plasma membrane Ca²⁺-ATPase (PMCA), NCX or mitochondria were selectively inhibited. In WT VSMC, NCX accounted for 90% of Ca²⁺ efflux after store release. In 2KO VSMC, preferential clearance of store released Ca²⁺ by NCX was lost, while PMCA activity was increased. Selective inhibition of the 2-isoform (0.5 µM ouabain for 20 minutes), prior to treatment with CPA, enhanced store load in VSMC from WT, but not 2KO mice. Subsequent analysis of capacitative Ca²⁺ entry (CCE) indicated that the magnitude of Ca²⁺ influx was significantly greater in 2KO cells. Our findings support the concept of a subsarcolemmal space where the 2-isoform coupled with NCX modulates Ca²⁺ store function, and thereby CCE.