Aortic stiffening is an independent predictor of negative cardiovascular outcomes and is associated with an increased risk of hypertension. Aortic fibrosis contributes to stiffening through the synthesis and deposition of collagen within the vessel wall. Endoplasmic reticulum (ER) stress has been linked with cardiac fibrosis and in non-vascular cell types ER stress can trigger collagen synthesis. However, the role of ER stress in aortic fibrosis and collagen synthesis remains unclear. We hypothesized that ER stress promotes aortic fibrosis through increasing collagen synthesis and deposition. In vitro, collagen synthesis was measured in the media from primary aortic vascular smooth muscle cells (VSMCs, third passage) treated at different time points (0, 6, 12, 24 hrs) with ER stress inducer, tunicamycin (TM, 1 μg/ml) using Sirius red and analyzed with a spectrophotometer (ODN 550 nM). In vivo, male Sprague-Dawley rats (12 wks) were implanted with mini-osmotic pumps containing TM (10 μg/kg/day, 14 days) or received sham surgery. Thoracic aorta was used for Western blot (data presented as fold change from control ± SEM) and immunohistochemical staining with Sirius red, a measure of collagen content and Masson’s trichrome stain, a measurement of fibrosis. Systolic blood pressure was increased in rats treated with TM compared to control (mmHg; 167±2 vs. 128±3). There was an increase in aortic wall thickness of the TM treated rats compared to control (mm; 0.35±0.01 vs. 0.32±0.01). Aortic expression of known proteins upregulated following induction of ER stress were increased by TM treatment: GRP78, an ER chaperone (1.4±0.3), PDI, a protein disulfide isomerase (1.3±0.2) and CHOP, a pro-apoptotic protein (1.7±0.3). Treatment with TM increased both aortic collagen content (% area; 10.7±0.5 vs. 7.4±0.4) and fibrosis (% area; 10.4±0.4 vs. 5.1±0.9) compared to control. There was a time-dependent increase in collagen synthesis from the media of VSMCs cells treated with TM (ODN 550nM: 0hrs, 0.9±0.002; 6hrs, 0.12±0.004; 12hrs, 0.17±0.005; 24hrs, 0.28±0.008). Our data suggest that ER stress may contribute to aortic stiffening through increasing collagen synthesis and deposition.