We used the human cerebral microvascular endothelial cells hCMEC/D3 to investigate whether the antithrombic drug dipyridamole affects gap junction coupling of blood-brain barrier (BBB) endothelial cells. Connexin37 (Cx37), Cx40, Cx43 and Cx45 were identified by PCR as putative expressed connexins in the cells. Scrape loading experiments showed that dipyridamole increased the gap junction coupling. A significant increase was already induced by 5 µM and was maximal at 25 µM. The increase was significant after an application time of 1 h and achieved a maximum after 9 h. PCR and Western blotting experiments did not show a correlation between enhancement of gap junction coupling and accumulation of connexin mRNA or protein, while immunocytochemical experiments showed an increased formation of Cx43 gap junction plaques between dipyridamole-treated cells. Together with the finding that brefeldin A did not inhibit the dipyridamole induced enhancement of gap junction coupling, the results suggest that dipyridamole affected connexons beyond the Golgi apparatus. As for the signaling pathway that was induced by dipyridamole, the PKA inhibitor Rp-cAMPS blocked the dipyridamole induced enhancement of gap junction coupling, while the adenosine receptor agonist PAA increased the gap junction coupling like dipyridamole. These pharmacological findings suggest that by acting as an inhibitor of adenosine transporters, dipyridamole induced a cAMP/PKA cascade which in return affected the gap junction coupling. Our results suggest that the beneficial effect of the antithrombic drug dipyridamole in prevention of secondary stroke might be related to the dipyridamole induced enhancement of gap junction coupling in the BBB endothelial cells.