Our previous studies have demonstrated that both the RhoA/Rho kinase and the protein kinase C (PKC) signaling pathways are involved in the low-dose endothelial monocyte-activating polypeptide-II (EMAP-II)-induced blood-tumor barrier (BTB) opening. In the present study, an in vitro BTB model was used to investigate which isoforms of PKC were involved in this process as well as the interactions between the RhoA/Rho kinase and the PKC signaling pathways. Our results showed that EMAP-II-activated PKC-α, β, and ζ and induced translocations of them from the cytosolic to the membrane fractions of rat brain microvascular endothelial cells. The EMAP-II-induced alterations in BTB permeability and tight junction (TJ) protein expression were partially blocked by GÖ6976, the inhibitor of PKC-α/β, and PKC-ζ pseudosubstrate inhibitor (PKC-ζ-PI). Meanwhile, we observed that GÖ6976 partly inhibited the EMAP-II-induced rearrangement of actin cytoskeleton as well as phosphorylation of myosin light chain and cofilin, whereas PKC-ζ-PI had no effect on these above-mentioned changes induced by EMAP-II. Also, our data revealed that inhibition of RhoA or inhibition of Rho kinase significantly diminished the activities and the translocations of PKC-α and PKC-β induced by EMAP-II, whereas PKC-ζ was unaffected. However, inhibition of PKC-α/β or inhibition of PKC-ζ did not cause any changes in the RhoA and Rho kinase activities. The effects of EMAP-II on BTB permeability and TJ proteins expression were completely blocked by inhibition of both RhoA and PKC-ζ, whereas inhibition of both RhoA and PKC-α/β had an effect similar to that of inhibition of RhoA alone. In summary, this study demonstrates for the first time that three PKC isoforms, PKC-α, β, and ζ, are involved in the EMAP-II-induced BTB opening. It is PKC-α/β, but not PKC-ζ, which serves as the downstream target for RhoA and Rho kinase, suggesting that EMAP-II induces BTB opening via the RhoA/Rho kinase/PKC-α/β signaling pathways. However, PKC-ζ is involved in this process by other mechanisms.