Vascular damage signals smooth muscle cells to proliferate, often exacerbating existent pathologies. Although the role of changes in global Ca²⁺ in vascular smooth muscle (VSM) cell dedifferentiation has been studied, the role that specific Ca²⁺ signals play in determining VSM phenotype remains relatively unexplored. Prior work with cultured VSM suggests that inositol-1,4,5-trisphosphate receptor (IP₃R) expression and sarcoplasmic reticulum (SR) Ca²⁺ release may be linked to VSM cell proliferation in native tissue. Thus, we hypothesized that SR Ca²⁺ release through IP₃Rs in the form of discrete transient signals is necessary for VSM proliferation. To investigate this, we designed an organ culture system using mouse cerebral arteries that permitted examination of Ca²⁺ dynamics in native tissue. Explanted arteries were cultured in normal media with 10% FBS and the appearance of individual VSM cells migrating from the explanted arteries (outgrowth cells) was tracked daily. Initial exposure to 10% FBS increased Ca²⁺ waves in myocytes in the arteries, which were blocked by the IP₃R antagonist, 2-aminethoxydiphenylborate (2-APB). Inhibition of IP₃R opening (via 100 µM 2-APB, 10 µM xestospongin C or 25 µM U73122) dramatically reduced outgrowth cell number in comparison to untreated or ryanodine-treated (10 µM) arteries. Consistent with this, 2-APB treatment inhibited cell proliferation, as measured by reduced PCNA immunostaining within 48 hours of culture, but did not inhibit cell migration. These results indicate that activation of IP₃R Ca²⁺ release is required for VSM cell proliferation in these arteries.