Arterial stiffness occurs when structural changes in the arterial wall, such as increased extracellular matrix (ECM) proteins or vascular smooth muscle cells (VSMCs), or cellular dysfunction disrupt the arteries' ability to adapt to changes in blood pressure or flow. This condition is linked to cardiovascular diseases and increased risks of adverse outcomes. Angiotensin II (AngII) contributes to arterial stiffness by constricting blood vessels, promoting VSMC proliferation, and enhancing ECM protein production. Scleraxis transcription factor contributes to cardiac fibrosis by upregulating ECM genes and activating fibroblasts to myofibroblasts. Scleraxis expression is upregulated in the high-pressure side of the aorta wall in the transverse aortic constriction (TAC) mouse model. In this project, we hypothesized that overexpression of scleraxis in fibroblasts or VSMCs using TCF21iCre or Myh11Cre approaches, respectively, would increase vascular stiffness in mesenteric arteries and aortas. We assessed mesenteric artery stiffness and function using pressure myography and performed immunohistology staining and gene expression analysis on harvested aortas. In vitro, we increased scleraxis expression in human aortic smooth muscle cells (HAOSMCs) and assessed proliferation capacity. Our findings showed that scleraxis upregulation in VSMCs decreased compliance in mesenteric arteries without altering the structure or function in these arteries. Scleraxis upregulation in HAOSMCs induced cell proliferation and a switch from a synthetic phenotype toward the proliferative phenotype. Mice with both scleraxis upregulation and AngII infusion exhibited stiffer mesenteric arteries. This was accompanied by a decrease in vascular lumen diameter, an increase in smooth muscle cell (SMC) contractility and impaired relaxation. Unexpectedly, the combination of scleraxis with AngII reduced ECM expression, suggesting arterial remodelling was occurring. HAOSMCs treated with AngII and scleraxis demonstrated a decrease in proliferation, and an increased expression of contractile genes, indicating a contractile VSMC phenotype. In summary, our findings suggest that upregulation of scleraxis expression in VSMCs alone induces vascular stiffness by promoting SMC proliferation. However, when combined with AngII, vascular stiffness is exacerbated by inducing a ' contractile' phenotype in SMCs. These findings highlight the complex and context-dependent role of scleraxis in vascular stiffness, emphasizing the importance of considering specific pathological conditions and factors when studying its effects on vascular function.