Exposure to polycyclic aromatic hydrocarbons (PAHs) contributes to the damage of blood-brain barrier. While a number of studies were focused on benzo[a]pyrene, direct effects and mechanisms of benzo[b]fluoranthene (B[b]F), another main component of PAHs, on blood-brain barrier (BBB) are not documented. Here, we investigated if B[b]F at concentrations of environmental relevance could affect apoptosis, oxidative stress, mitochondrial membrane potential (MMP) and BBB marker expression in mouse brain microvascular endothelial (bEnd.3) cells, an in vitro model typically used to study BBB toxicology. Cells were treated with varying concentrations of B[b]F (0, 10, 20 and 40 μM) for 48 h. Cell proliferation, cell cycle, apoptosis, oxidative stress, MMP and BBB marker expressions were evaluated by label-free real-time cell analysis, flow cytometry, immunofluorescence and Western-blot. The proliferation of bEnd.3 cells was inhibited by B[b]F in a concentration dependent manner. B[b]F treatment significantly affected cell cycle, induced apoptosis, increased levels of reactive oxygen species (ROS) and disputed MMP. Expressions of BBB marker Occludin and Claudin-5 were decreased in the presence of 40 μM B[b]F. In conclusion, B[b]F might damage BBB by affecting proliferation, apoptosis, ROS level and Occludin and Claudin-5 expressions in microvascular endothelial cells.