Inflammation-induced vascular endothelial dysfunction can allow plasma proteins to cross the vascular wall causing edema. Proteins may traverse the vascular wall through two main paracellular and transcellular transport pathways. Paracellular transport involves changes in endothelial cell (EC) junction proteins, while transcellular transport involves caveolar transcytosis. Since both processes are associated with filamentous actin formation the two pathways are interconnected. Therefore, it is difficult to differentiate the prevailing role of one or the other pathways during various pathologies causing an increase in vascular permeability. Using a newly developed dual-tracer probing method, we differentiated transcellular from paracellular transport during conditions of increased content of fibrinogen (Fg), called hyperfibrinogenemia (HFg). Roles of cholesterol and sphingolipids in formation of functional caveolae were assessed using a cholesterol chelator, methyl-beta-cyclodextrin (MβCD) and the de novo sphingolipid synthesis inhibitor myriocin. Fg-induced formation of functional caveolae was defined by association and co-localization of sodium-potassium ATPase (Na⁺/K⁺-ATPase) and plasmalemmal vesicle-associated protein-1 (PV-1) using Försters' Resonance Energy Transfer (FRET) and Total Internal Reflection Fluorescence (TIRF) microscopies, respectively. HFg increased permeability of the EC layer mainly through the transcellular pathway. While MβCD blocked Fg-increased transcellular and paracellular transports, myriocin affected only the transcellular transport. Pial venular leakage of albumin was lesser in myriocin-treated HFg mice. HFg induced greater formation of functional caveolae as indicated by co-localization of Na⁺/K⁺-ATPase with PV-1 by FRET and TIRF. Our results suggest that elevated blood levels of Fg alter cerebrovascular permeability mainly by affecting caveolae-mediated transcytosis through modulation of de novo sphingolipid synthesis.