Ultrasound-targeted microbubble cavitation (UTMC) transiently opens the blood brain barrier (BBB). We previously determined that UTMC induces BBB hyperpermeability through an influx of calcium. As activation of RhoA is a calcium-dependent pathway that causes cytoskeletal reorganization, leading to the breakdown of tight junctions, we tested the hypothesis that UTMC-induced activation of RhoA leads to BBB hyperpermeability. We utilized a transwell model with brain endothelial cells and astrocytes on opposite sides of a support membrane. Ultrasound (1 MHz, 250 kPa, 10 μs pulse duration, 10 ms pulse interval) was applied in the presence of lipid microbubbles for 20 s. BBB permeability was assessed using dextran flux and transendothelial electrical resistance (TEER) measured across the membrane. Integrity of tight junctions was evaluated by staining for ZO-1. UTMC reduced TEER (p < 0.05), confirming reduced barrier integrity. One hour after UTMC, there was a significant decrease in ZO-1 mean pixel intensity (p < 0.05). Treatment of cells with Rho inhibitor II (Y16) significantly reduced UTMC-induced dextran flux across the BBB (p < 0.05) and UTMC-induced decrease in TEER. In our co-culture model of the BBB, UTMC induces hyperpermeability through modulation of tight junctions, at least in part through a RhoA-dependent mechanism.