Maintenance of single-layered endothelium, squamous endothelial cell shape, and formation of a patent vascular lumen all require defined endothelial cell polarity. Loss of β1 integrin (Itgb1) in nascent endothelium leads to disruption of arterial endothelial cell polarity and lumen formation. The loss of polarity is manifested as cuboidal-shaped endothelial cells with dysregulated levels and mislocalization of normally polarized cell-cell adhesion molecules, as well as decreased expression of the polarity gene Par3 (pard3). β1 integrin and Par3 are both localized to the endothelial layer, with preferential expression of Par3 in arterial endothelium. Luminal occlusion is also exclusively noted in arteries, and is partially rescued by replacement of Par3 protein in β1-deficient vessels. Combined, our findings demonstrate that β1 integrin functions upstream of Par3 as part of a molecular cascade required for endothelial cell polarity and lumen formation.