AbstractAtherosclerotic plaques are commonly observed at low shear regions, in particular, the left main coronary artery (LMCA) bifurcation. Low shear regions at bifurcations promote endothelial dysfunction, a key factor initiating atherogenesis, however, mechanisms underlying this process are poorly understood. Dynamic in vitro models are critical to investigate endothelial dysfunction, but current static and vessels‐on‐chip systems typically lack physiologically complex geometries and local shear changes. Here, a bifurcating coronary artery‐on‐a‐chip is developed, mimicking the human LMCA, displaying reduced shear near the bifurcation, verified using computational fluid dynamics simulations. Over 7 days of dynamic culture, human coronary artery endothelial cells aligned with the flow and expressed more Endothelial nitric oxide synthase (eNOS) and intercellular cell adheison molecule‐1 (ICAM‐1) at high shear regions (12.7 dyn cm−2) adjacent to, but not at the bifurcation (0–3 dyn cm−2). After tumor necrosis factor‐alpha (TNFα) stimulation to induce endothelial dysfunction, spatially mapping cellular changes and shear gradients revealed cell alignment is disrupted over a larger area surrounding the bifurcation at a higher shear, and ICAM‐1 expression is increased closer to the bifurcation at a lower shear. This coronary artery‐on‐a‐chip establishes a system to spatially map endothelial cell behavior in response to differential shear and vessel geometry, enabling future studies into plaque initiation events, treatment targets, and drug screening.