Endothelial cytoplasmic calcium ([Ca²⁺]_i) is an important factor determining blood-brain barrier (BBB) permeability but little is known on the influence of [Ca²⁺]_i dynamics on BBB function. Here, we applied several conditions that trigger intercellular Ca²⁺ waves or intracellular oscillations and determined the involvement of connexin (Cx) channels and consequent effects on BBB function, making use of RBE4 and primary capillary endothelial cells. Exposure to low extracellular Ca²⁺ or bradykinin (BK) respectively triggered Ca²⁺ waves or oscillations that increased BBB permeability in a [Ca²⁺]_i-dependent manner. Both Ca²⁺ waves/oscillations and BBB alterations were inhibited by the Cx mimetic peptide Gap27. Ca²⁺ wave propagation involves gap junctional communication and opening of hemichannels but hemichannels did not contribute as a BBB permeability-increasing pathway. Ca²⁺ oscillations were inhibited by Cx37/43 knockdown and involved hemichannel opening as well as autocrine purinergic signaling, but hemichannels were, as for Ca²⁺ waves, not the pathway of increased BBB permeability. Exposure to ATP triggered, like BK, Ca²⁺ oscillations but in contrast, these were not affected by Gap27 and did not disturb BBB function. We conclude that Cx channels and purinergic signaling contribute to endothelial [Ca²⁺]_i dynamics and are essential in altering BBB function.