The blood-brain barrier (BBB) consists in part of a highly specialized set of cells which separates the brain from the vascular system. The BBB controls the entry and exit of substances from the brain tissue through tight junctions (TJs) between endothelial cells. It is known that the hormone prolactin (PRL) is able to regulate endothelial-dependent processes, like the balance between proliferation and apoptosis and the mammary epithelial permeability. However, the effects of PRL and the role it plays in the BBB permeability are still not well understood. A primary culture of bovine brain microvessel endothelial cells was used as in vitro model of BBB. Cells were treated with PRL (0.1, 1, 10 and 100 nM) for 24 hours. PRL significantly increased cellular proliferation at 10 and 100 nM, but did not modify basal apoptosis. These effects were dependent on the production of the mitogenic factor nitric oxide (NO). PRL significantly decreased the permeability and promoted an increase in trans-endothelial electrical resistance in a NO-independent way. PRL also increased the expression of the TJs proteins claudin-5 and occludin. The short form of the PRL receptor was detected in these cells but its expression was not modified by PRL. Together, these results suggest that PRL has the ability to increase cellular proliferation associated with a decrease on BBB permeability by increasing the expression of TJs proteins.