BACKGROUND & PURPOSE: Arterial stiffness, or loss of elastic compliance in large arteries, is an independent precursor of cardiovascular disease (CVD) [1] and dementia [2] for which currently there are no targeted therapies. We previously discovered that decreases in NO-sensitive guanylyl cyclase (NO-GC), the NO receptor which synthesizes cGMP, and in its target vasodilator-stimulated phosphoprotein (pVASP), lead to increased cytoskeletal actin polymerization in vascular smooth muscle cells (VSMCs) contributing to increased arterial stiffness [3]. In the current study, we tested whether activating NO-GC with an NO-GC activator (cinaciguat) modulates pVASP and cytoskeletal actin polymerization in VSMCs, thereby preventing obesity-induced arterial stiffness. EXPERIMENTAL APPROACH & KEY RESULTS: Cinaciguat administration (5 mg/kg) to high fat, high sucrose diet (HFHS)-fed mice, our established model of arterial stiffness [4], (1) decreased pulse wave velocity, the in vivo index of arterial stiffness, without affecting blood pressure; (2) increased aortic pVASP levels; and (3) decreased actin polymerization, measured as ratio of filamentous (F) to globular (G) actin, compared to vehicle administration. In cultured VSMCs, cinaciguat (10 μmol/L) increased pVASP levels and decreased the F/G actin ratio at baseline and after stimulation with the cytokine tumor necrosis factor α (TNFα), which we previously showed is significantly increased in the aorta of HFHS-fed mice [4-6]. These effects were abrogated in aortas and VSMCs from mice with smooth muscle-specific cGKI deletion (cGKI), while being mimicked by a cell-permeable cGMP analog (8-Br-cGMP), which also decreased VSMC stiffness in vitro. CONCLUSIONS & IMPLICATIONS: Collectively, our data strongly support the notion that pharmacological NO-GC activation would be beneficial in decreasing obesity-associated arterial stiffness by decreasing VSMC cytoskeletal actin hyper-polymerization. If translated to humans, NO-GC activators could become a viable approach to clinically treat arterial stiffness, which remains an unmet medical need.