Abstract Body: Cardiac hormones atrial and brain natriuretic peptides (ANP and BNP) regulate blood pressure, volume homeostasis, and exert their biological actions by binding to the natriuretic peptide receptor-A (NPR-A), resulting in the generation of the second messenger cGMP. NPRA is expressed in various tissues and cells, including the vascular smooth muscle cells and endothelial cells. However, the role of ANP/NPRA signaling in smooth muscle cells (SMCs) remains unclear. This study aimed to determine the effect of conditional deletion of SMC-specific Npr1 (encoding NPRA) gene-disrupted male mice. The wild-type control (SMC-Npr1 f/f; WT), heterozygous (SMC-Cre-Npr1 f/+; HT), and knockout (SMC-Cre-Npr1 f/-; KO) male mice were intraperitoneally injected with tamoxifen (20 mg/day in Olive oil) on 5 consecutive days to inactivate Npr1 followed by a 10-day off period, after which all experiments were commenced. Systolic blood pressure (SBP) was determined by non-invasive computerized tail-cuff method (Visitech 2000), carotid-femoral pulse wave velocity (cfPWV) and intracarotid PWV (icPWV) were assessed with high resolution ultrasound. Endothelium denuded aortic rings reactivity was measured using wire myography in male SMC-specific Npr1 disrupted HT, KO, and WT mice. The nuclear factor-kappa B 65 (NF-kB p65) and toll-like receptors 2/4 (TLR2, TLR4) proteins in the endothelium-denuded aortic rings were measured by western blot. SBP was significantly (P < 0.01; P < 0.001) elevated in SMC-Npr1 HT (114 ± 2 mmHg) and KO mice (131 ± 3 mmHg) compared to WT (101 ± 2 mmHg) mice. Mean arterial pressure (MAP) measured by telemetry, was increased by 26+2 mmHg in SMC-Npr1 KO mice compared with SMC-Npr1 WT mice. cfPWV and icPWV were significantly (P < 0.05; P < 0.01) increased in SMC- Npr1 KO mice compared to WT mice. Treatment with increasing concentrations of ANP (IC50 = 6 × 10^-9M) relaxed aortic rings that had been contracted with PGF2α. However, the relaxation activity in the aortic rings of SMC-Npr1 KO mice was significantly lower than that of WT mice. The NF-kB p65, TLR2, and TLR4 protein expression were markedly increased in SMC- Npr1 HT and SMC-Npr1 KO mice compared to WT mice. In summary, the present findings suggest that in SMC, ANP/NPRA signaling may play a crucial role in maintaining blood pressure and regulating vasorelaxation activity and inflammation. Supported by NIH/NIDDK grant (DK133833).