Objective This study was performed with an in vitro blood-tumor barrier (BTB) model to investigate whether protein kinase C (PKC) and RhoA are involved in the low-dose endothelial monocyte-activating polypeptide-Ⅱ (EMAP-Ⅱ)-induced BTB opening.Methods The permeability of BTB model was detected by transendothelial electric resistance (TEER) and Horseradish peroxidase (HRP) assays.The total PKC activity was measured by using PKC Assay kit.Western blot assay was used to test the protein expression of total PKC, PKC isoforms (α, β, γ, ε, δ, and ζ), Rho A and tight junction (TJ)-associated proteins (occludin, cluadin-5 and ZO-1).The distribution and expression changes of TJ associated proteins and cytoskeleton protein (F-actin) were tested by immunofluorescence assays.Results In BMECs treated with EMAP-Ⅱ (0.05 nmol/L) for 0.5 h and 1 h, the significant increases in total PKC activity and the protein expression of total PKC were observed.Meanwhile, the protein expression of 3 PKC isoforms (PKC-α, β and ζ) and RhoA significantly increased at 0.5 h and 1 h after EMAP-Ⅱ administration and then recovered at 4 h.TEER and HRP flux assays revealed that pretreatment with PKC inhibitor H7, RhoA inhibitor C3 exoenzyme or a combination of the two inhibitors significantly blocked the EMAP-Ⅱ-induced BTB permeability change.Moreover, our data showed that the EMAP-Ⅱ-induced downregulation of occludin, claudin-5, ZO-1 as well as F-actin was also significantly inhibited by pretreatment with H7, C3 exoenzyme or a combination of H7 and C3 exoenzyme.Conclusion In summary, our present study suggests that PKC and RhoA play crucial roles in the process of EMAP-Ⅱ inducing progressive changes to BMECs of BTB, including downregulation of tight junction proteins, reorganization of actin cytoskeleton, and increasing of BTB permeability.