Arterial stiffness is a risk factor for several cardiometabolic diseases and is caused by pathological remodeling of the vascular extracellular matrix (ECM). Vascular smooth muscle cells (VSMCs) respond to ECM stiffness by proliferating, migrating, and further remodeling the vascular ECM, thus contributing to vascular disease like atherosclerosis and hypertension. VSMCs along the vasculature are highly diverse as they arise from different embryologic origins, reside in ECMs of diverse compositions, and are exposed to various mechanical forces. This dissertation aims to understand how ECM stiffness regulates the transcriptional response of VSMCs from different origins, namely aortic (Ao) and coronary (Co) VSMCs. We conducted deep sequencing of RNA from Ao and Co VSMCs grown on engineered polyacrylamide hydrogel surfaces tuned to physiologic and pathologic stiffness. Using several bioinformatic approaches, we compared the transcriptional landscapes in Ao and Co VSMCs by looking at whole-gene level expression, splicing, and conservation properties, with a focus on long non-coding RNAs (lncRNAs), as they compose a significant portion of the unexplored VSMC transcriptome. We found evidence suggesting that the overall transcriptional response to stiffness may be conserved across species and cell types, and that stiffness significantly dictates VSMC transcriptional identity over contributions from embryologic origins. However, we also discovered instances of origin-specific stiffness responses in stiffness-mediated lncRNA expression and stiffness-mediated splicing. We identified a highly correlated network of adjacent stiffness-sensitive lncRNAs-protein coding gene pairs, which led us to experimentally interrogate the lncRNA PACER as a regulator of stiffness-mediate expression. We surprisingly found that PACER’s previously established regulatory pathway is absent in VSMCs. Using enrichment methods, we identified TBX5 and show that it is a stiffness-sensitive transcription factor specific to Co VSMCs. We also demonstrated a new role for MALAT1 as a lncRNA regulator of stiffness-dependent VSMC proliferation and migration. Thus, this dissertation reveals many novel characteristics of the VSMC stiffness-regulated transcriptome that may have clinical utility in understanding and managing the pathogenesis of arterial stiffening.