The polarized functions of epithelia require an intact tight junction (TJ) to restrict paracellular movement and to separate membrane proteins into specific domains. TJs contain scaffolding, integral membrane and signaling proteins, but the mechanisms that regulate TJs and their assembly are not well defined. Galpha12 (GNA12) binds the TJ protein ZO-1 (TJP1), and Galpha12 activates Src to increase paracellular permeability via unknown mechanisms. Herein, we identify Src as a component of the TJ and find that recruitment of Hsp90 to activated Galpha12 is necessary for signaling. TJ integrity is disrupted by Galpha12-stimulated Src phosphorylation of ZO-1 and ZO-2 (TJP2); this phosphorylation leads to dissociation of occludin and claudin 1 from the ZO-1 protein complex. Inhibiting Hsp90 with geldanamycin blocks Galpha12-stimulated Src activation and phosphorylation, but does not affect protein levels or the Galpha12-ZO-1 interaction. Using the calcium-switch model of TJ assembly and GST-TPR (GST-fused TPR domain of PP5) pull-downs of activated Galpha12, we demonstrate that switching to normal calcium medium activates endogenous Galpha12 during TJ assembly. Thrombin increases permeability and delays TJ assembly by activating Galpha12, but not Galpha13, signaling pathways. These findings reveal an important role for Galpha12, Src and Hsp90 in regulating the TJ in established epithelia and during TJ assembly.