Studies have utilised immortalised mouse cerebral endothelial cells (bEnd.3) exposed to oxygen glucose deprivation (OGD) to study blood–brain barrier (BBB) disruption after ischaemia. However, there is a paucity of literature describing the duration of OGD (and reoxygenation [RO]) required to best simulate BBB disruption in vivo. In this study we assessed BBB disruption in bEnd.3 cells after exposure to a range of OGD periods, and also after OGD + RO. Exposure of bEnd.3 monolayers to 4, 6, 16, or 24 hours of OGD resulted in a significant increase in permeability. The hyperpermeability after 16 or 24 hours was associated with decreased expression of tight junction proteins (occludin and claudin‐5). Furthermore, there was a decrease in cell viability and increased expression of the pro‐apoptotic protein, cleaved caspase‐3. Exposure of bEnd.3 monolayers to 1 hour OGD+ 23 hours RO exacerbated hyperpermeability relative to 1 hour OGD, which was associated with decreased expression levels of occludin and ZO‐1, but no change in cell viability or caspase‐3. 4 hours OGD + 23 hours RO exacerbated hyperpermeability, decreased expression levels of tight junction proteins, decreased cell viability, and increased caspase‐3 expression. Thus, bEnd.3 cells exhibit hyperpermeability, a loss of tight junction proteins, and undergo cell death, after exposure to prolonged periods of OGD. Moreover, they exhibit exacerbated hyperpermeability, a loss of tight junction proteins, and increased expression of caspase‐3 after OGD + RO. These findings will facilitate the use of this cell line in studies of BBB disruption and for the testing of therapeutics.