Abstract: Objective Here we studied regulation of VSMC proliferation by NO/PKG mediated via modulating intracellular Ca2+/calcineurin (CaN) signaling pathway. Methods Primary VSMCs from rat aorta were used as the experimental model. CaN protein and its activity were assayed using immunoblotting and free inorganic phosphate content analysis, respectively. growth and survival rate of cells were determined by MTT assay and acridine orange and ethidium bromide staining. Results The addition of SNAP and Sp-8-pCPT-cGMPS decrease absorbance of cells stimulated by phenylephrine (PE), whereas the addition of Rp-8-pCPT-cGMPS increases it. In SMCs pretreated with Ver, absorbance of cells stimulated by PE decreased and was further inhibited by the additional treatment of SNAP and Sp-8-pCPT-cGMPS. In SMCs pretreated with CsA, absorbance of cells stimulated by PE decreased, but it could not be further altered by the additional treatment of SNAP, Sp-8-pCPT-cGMPS and Rp-8-pCPT-cGMPS. Moreover, expression and activities of CaN induced by PE was inhibited by SNAP and Sp-8-pCPT-cGMPS. Conclusions NO/PKG inhibits the proliferation of vascular SMCs without decreasing cell survival rate, which is mediated via intracellular Ca2+/CaN signaling pathway.