Inhaled atmospheric nanoparticles (ANPs) can migrate into human blood vessels. However, the exact pathogenesis has not yet been well elucidated. In this study, a perfusable 3D human microvessel network was constructed in a microfluidic device. This functional 3D micro-tissue partly mimicked the physiological response of human vessels. Intravascular nanoparticles tend to adsorb proteins to form a protein corona. Based on this pathological response, vessel permeability and vasoconstriction resulting from ANP stimulation might be related to vascular inflammation. It mediated abnormal expression of nuclear factor-κB (NF-κB) and an influx of intracellular Ca2+ ([Ca2+]i). This biological behavior disturbed the normal expression of intercellular cell adhesion molecule 1 (ICAM-1) and vascular endothelial growth factor (VEGF). The imbalance of nitric oxide (NO) and endothelin-1 (ET-1) further resulted in endothelial cell contraction. All these bio-events induced the loss of tight junctions (ZO-1) which enhanced vessel permeability. Meanwhile, ANP induced-vascular toxicity was also found in mice. Our observations provide a plausible explanation for how the ANPs affect human vascular function. The vessel-on-chip provides a bridge between in vitro results and human responses. We aimed to use this human 3D functional microvascular model to mimic the physiological responses of human vessels. This model is suitable for the evaluation of vascular toxicity after the human vessel exposure to ANPs.