The calcium-activated potassium channel (K (Ca) channel) activator, NS1619, has been shown to selectively and time-dependently increase the permeability of the blood-tumor barrier (BTB) by downregulating the expression of tight junction (TJ) protein. However, the role of signaling cascades in this process has not been precisely elucidated. This study was performed to determine the role of signaling cascades involving reactive oxygen species (ROS)/RhoA/PI3K/PKB in increasing the permeability of the BTB induced by NS1619. Using an in vitro BTB model and selective inhibitors of signaling pathways, we investigated whether ROS/RhoA/PI3K/PKB pathway plays a key role in the process of the increase in BTB permeability induced by NS1619. The results revealed that the BTB permeability was increased and the expression of TJ proteins were significantly decreased by NS1619, and selective inhibitors of identified signaling pathways reversed the observed alterations. Moreover, the significant increases in ROS, RhoA activity, and PKB phosphorylation after NS1619 administration were observed, which were partly inhibited by N-2-mercaptopropionyl glycine or C3 exoenzyme or LY294002 pretreatment. The present study demonstrates that the activation of signaling cascades involving ROS/RhoA/PI3K/PKB in rat brain microvascular endothelial cells was required for the increase in BTB permeability induced by NS1619.