The purposes of this study were to characterize the direct effect of the C-terminal fragment of fibrinogen gamma chain (gammaC) on microvascular endothelial permeability and to examine its molecular mechanism of action. Intravital microscopy was performed to measure albumin extravasation in intact mesenteric microvasculature, followed by quantification of hydraulic conductivity in single perfused microvessels. Transendothelial electric resistance was measured in microvascular endothelial cells in combination with immunoblotting and immunocytochemistry. The results show that gammaC induced time- and concentration-dependent increases in protein transvascular flux and water permeability and decreases in endothelial barrier function, coupled with Rho GTPase activation, myosin light chain phosphorylation, and stress fiber formation. Depletion of RhoA via siRNA knockdown or pharmacological inhibition of RhoA signaling attenuated gammaC-induced barrier dysfunction. Imaging analyses demonstrated binding of gammaC to endothelial cells; the interaction was inhibited during blockage of the alphavbeta3 integrin. Furthermore, in vivo experiments showed that the microvascular leak response to gammaC was attenuated in integrin beta3(-/-) animals. Fibrinogen-gamma C terminus directly interacts with the microvascular endothelium causing fluid and protein leak. The endothelial response to gammaC involves an integrin receptor-mediated RhoA-dependent signaling pathway that leads to paracellular hyperpermeability.