Abstract Body: Arterial stiffness, a hallmark of impaired vessel tone, contributes to elevated blood pressure (BP) and cardiovascular risk. Our prior work showed that RhoBTB1 regulates BP, nitric oxide-dependent tone and arterial stiffness by targeting Phosphodiesterase 5 for CUL3-mediated proteasomal degradation. But, the full spectrum of RhoBTB1 protein targets remains unknown. We used ascorbate peroxidase (APEX2) proximity labeling and mass spectrometry to profile the A7R5 smooth muscle cell (SMC) proteome and identified 59 potential RhoBTB1-interacting proteins. Herein, we focused on RNA-binding motif protein 9 (RBM9/RbFox2), a key regulator of RNA splicing. Co-immunoprecipitation (Co-IP) confirmed the interaction between RbFox2 and RhoBTB1. RbFox2 expression was elevated in response to proteasome (MG132) and CUL inhibition (MLN4924), and in CUL3-deficient HEK293 cells, SMC-specific CUL3-deficient mouse aortas, RhoBTB1 knockdown in A7R5 cells, and angiotensin-II (ANG). siRNA-mediated silencing of RbFox2 in A7R5 cells and deletion of RbFox2 in primary aortic SMCs altered the organization of actin as measured by phalloidin staining. RNA-Seq of RbFox2-deficient A7R5 cells also suggested transcriptional downregulation of genes encoding cytoskeletal components. We hypothesized that RbFox2 regulates the actin cytoskeleton and contributes to vascular stiffness, and that its deletion in SMCs (S-RbFox2KO) could alleviate ANG-induced arterial stiffness. To investigate this, mice carrying a conditional allele of RbFox2 (RbFox2Flox) were bred with mice carrying tamoxifen (Tx)-inducible CRE-recombinase under a smooth muscle promoter (SMC-CRE). Hypertension and arterial stiffness were induced using ANG (490 ng/kg/min for 3 weeks), and both were measured weekly. Both groups had equivalent SBP (109.5±3.06 vs 110.2±3.01 mmHg; S-RbFox2KO vs SMC-CRE) and pulse wave velocity (PWV, 2.7±0.08 vs 2.63±0.07 mm/ms; S-RbFox2KO vs SMC-CRE) at baseline. 3-weeks of ANG increased BP (142.29±3.22 vs 135.44±6.26) and PWV (3.84±0.08 vs 3.93±0.06) in both groups. Tx was then administered with continued ANG treatment. Post-Tx, BP increased in both groups, but the difference was not significant (148.1±2.14 vs 156.1±4.25). Whereas PWV increased in SMC-CRE mice (4.52±0.17), it remained stable in S-RbFox2KO mice (4.0±0.19). These findings strongly suggest that RbFox2 is regulated by the RhoBTB1-CUL3 axis and modulates arterial stiffness through modulating the cytoskeleton.