The blood-brain barrier (BBB) is a metabolic and physiological barrier important for maintaining brain homeostasis. The aim of this study was to determine the role of PKC activation in BBB paracellular permeability changes induced by hypoxia and posthypoxic reoxygenation using in vitro and in vivo BBB models. In rat brain microvessel endothelial cells (RMECs) exposed to hypoxia (1% O₂-99% N₂; 24 h), a significant increase in total PKC activity was observed, and this was reduced by posthypoxic reoxygenation (95% room air-5% CO₂) for 2 h. The expression of PKC-βII, PKC-γ, PKC-η, PKC-μ, and PKC-λ also increased following hypoxia (1% O₂-99% N₂; 24 h), and these protein levels remained elevated following posthypoxic reoxygenation (95% room air-5% CO₂; 2 h). Increases in the expression of PKC-ε and PKC-ζ were also observed following posthypoxic reoxygenation (95% room air-5% CO₂; 2 h). Moreover, inhibition of PKC with chelerythrine chloride (10 μM) attenuated the hypoxia-induced increases in [¹⁴C]sucrose permeability. Similar to what was observed in RMECs, total PKC activity was also stimulated in cerebral microvessels isolated from rats exposed to hypoxia (6% O₂-94% N₂; 1 h) and posthypoxic reoxygenation (room air; 10 min). In contrast, hypoxia (6% O₂-94% N₂; 1 h) and posthypoxic reoxygenation (room air; 10 min) significantly increased the expression levels of only PKC-γ and PKC-θ in the in vivo hypoxia model. These data demonstrate that hypoxia-induced BBB paracellular permeability changes occur via a PKC-dependent mechanism, possibly by differentially regulating the protein expression of the 11 PKC isozymes.