Abstract Body: Introduction: Nicotine, a key component of cigarette smoke, exacerbates atherosclerosis by promoting endothelial-to-mesenchymal transition (EndoMT) in endothelial cells (ECs). However, the interaction between nicotine-induced EndoMT and the extracellular matrix (ECM) properties, such as stiffness and protein composition, is poorly understood due to limitations in current models. Methods: We engineered an Atherosclerosis-on-a-Chip platform comprising a microarray of ECM components, including collagen types II, III, and IV, fibronectin, and laminin, under controlled stiffness conditions ranging from 150 kPa to 900 kPa. Human ECs were seeded onto the ECM arrays, and inflammatory cytokines (TNF-α, IFN-γ, oxLDL) were administered to mimic an atherosclerotic environment. Nicotine was subsequently introduced 6 days to analyze its impact on EndoMT markers (SM22α and VE-cadherin) across different ECM compositions and stiffness levels. Results: Our results show the contribution of nicotine in exacerbating the incidence of EndoMT, along with the role of nicotine interactions with ECM biochemistry and matrix stiffness in mediating the process. Among 32 unique conditions that encompass a range of stiffnesses and ECM protein combinations, our results show that about half of them show an increase in the incidence of EndoMT in the presence of nicotine. In particular, at the pathological stiffnesses of 150 kPa, ECMs comprising collagen IV alone or in combination with other ECMs (fibronectin or collagen II) showed the largest fold-change increase in EndoMT in the presence of nicotine, based on the immunofluorescence staining intensity of SM22a. However, in some ECM formulations, such as the combination of collagen II and laminin, this effect was not significant. Interestingly, in certain conditions, such as collagen II and collagen III at 150 kPa, nicotine even reversed the effect, reducing the mesenchymal transition. Conclusion: Nicotine promotes EndoMT, with its effects enhanced by stiffer ECM and mitigated by specific ECM compositions at lower stiffness. These findings highlight the significance of ECM environment in modulating cellular responses to nicotine and inflammatory mediators in this atherosclerosis-on-a-chip platform.