We hypothesized that human smoking and its deleterious effects on endothelial function can be modeled by exposure of mice to tobacco smoke, and further that these changes would be reflected in gene regulation in vascular endothelium. We used for these studies a mouse strain that expresses green fluorescent protein under the control of an endothelial-specific promoter, Tie-2. Mice were exposed to side-stream smoke from reference cigarettes at 34 mg total suspended particulates/m³. After exposure for 5 days/week for 1 and 6 weeks, aortae were pooled from treatment and control groups. Endothelial cells were rapidly isolated by collagenase treatment followed by fluorescent activated cell sorting to yield populations of >95% purity. RNA isolated from >500 endothelial cells was amplified and analyzed on deeply representative long oligo microarrays. Transcripts dysregulated by > 2.5-fold were confirmed by real-time PCR and selected proteins by immunofluorescent localization. In the endothelial cells, the observed >3-fold upregulation of Cfh (complement factor H/adrenomedullin binding protein), Calcr1 (calcitonin receptor-like) and Epxh2 (soluble epoxide hydrolase) may play a role in hypertensive responses of the vasculature to smoking. We have identified gene regulation in vivo in vascular endothelium that potentially underlies hypertensive responses to tobacco smoke.