Brain microvascular endothelial cells are connected by tight junction (TJ) proteins and interacted by adhesion molecules, which participate in the selective permeability of the blood-brain barrier (BBB). The disruption of BBB is associated with the progression of cerebral diseases. Pterostilbene is a natural compound found in blueberries and grapes with a wide range of biological activities, including anti-inflammatory, antioxidant, and anti-diabetic effects. In this study, we investigated the protective effects of pterostilbene on LPS-stimulated mouse brain endothelial (bEnd.3) cells and underlying mechanisms. The results showed that pterostilbene effectively upregulated the expressions of tight junction (TJ) proteins such as zonula occludens (ZO)-1 and claudin-5 and downregulated the expression of adhesion molecules such as intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1, preventing BBB damage under LPS stimulation. Pterostilbene decreased the LPS-triggered expressions of inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 as well as the levels of interleukin (IL)-6, tumor necrosis factor (TNF)-α and nitric oxide (NO). Meanwhile, we found that pterostilbene exerted an inhibitory effect on nuclear factor kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) pathways in bEnd.3 cells upon LPS stimulation. Additionally, pterostilbene exhibited antioxidant effects by activating heme oxygenase 1 (HO-1). These findings indicated that pterostilbene protected against lipopolysaccharide (LPS)-induced inflammation, oxidative stress and blood-brain barrier (BBB) disruption in bEnd.3 cells.