Abstract Body: Background: Clinical evidence suggests a greater impact of arterial stiffness on cardiovascular mortality in women vs. men. Human single nucleotide polymorphisms in the gene encoding for estrogen receptor α (ERα) associate with protection from arterial stiffness in post-menopausal women compared with age-matched men. We therefore hypothesized that ERα, specifically in smooth muscle cells (SMC), contributes to sex differences in arterial stiffening. Methods: We created novel SMC-specific ERα knockout (SMC-ERα-KO) mice to assess the role of SMC-ERα in sex-related differences in arterial stiffness. Primary outcomes included aortic pulse wave velocity (PWV, an index of arterial stiffness), histological assessment of aortic fibrosis and elastin degradation (Masson’s trichrome and Verhoeff van Gieson staining, respectively), aortic protein expression, and intrinsic SMC stiffness measured via atomic force microscopy. Outcomes were measured in 3- and 18-month-old female and male, SMC-ERα-intact and KO mice. Results: There was an aging-associated increase in PWV in SMC-ERα-intact female (p<0.0001) and male (p<0.0001) mice. The aging-associated increase in PWV was fully prevented in SMC-ERα-KO females (p<0.0001) and partially prevented in SMC-ERα-KO males (p<0.0001), such that PWV in aged SMC-ERα-KO males was still greater than young males of either genotype (both p<0.0001). There was an aging-associated increase in aortic fibrosis in female and male mice (both p<0.001) that persisted regardless of the deletion of SMC-ERα. There was an aging-associated increase in aortic elastin degradation in aortas from female (p<0.0001) and male mice (p<0.01). However, in female mice only, there was reduced elastin degradation in SMC-ERα-KO mice (p<0.04), while the deletion of SMC-ERα had no effect in males (p=0.35). In female mice, aortic expression of matrix metalloproteinase 2 followed the same pattern as histological elastin degradation (increase with aging: p<0.0001, decrease with SMC-ERα-KO: p=0.04). The deletion of SMC-ERα had no effect on intrinsic SMC stiffness in young or aged female or young male mice. However, in aged male mice, the deletion of SMC-ERα resulted in lower intrinsic SMC stiffness (p=0.03) compared with SMC-ERα-intact males. Conclusion: These data demonstrate that SMC-ERα contributes to aging-associated arterial stiffening in female and male mice via sex-specific mechanisms, with SMC-ERα promoting elastin degradation in females and SMC stiffness in males.