Background: Previous study showed that an aqueous extract of Bixa orellana L. (Family of Bixaceae) leaf (AEBO) is capable of inhibiting bradykinin (BK)-induced inflammation in animal models. Objective: This study further investigates the effect of AEBO on BK-induce inflammation in vitro model. Materials and Methods: The endothelial barrier protective effect of AEBO was examined via an in vitro endothelial permeability assay. Human umbilical vein endothelial cell (HUVEC) was first pretreated with AEBO with a concentration range from 0.1, 0.2, and 0.4 mg/mL and then induced with BK. Fluorescein isothiocyanate-conjugated-dextran was used as an indicator of permeability flux. To elucidate its mechanism of action, the phospholipase C (PLC) – nitric oxide (NO) – cyclic guanosine monophosphate (cGMP) signaling pathway and protein kinase C (PKC) activity were evaluated. Results: Pretreatment of AEBO significantly (P < 0.05) suppressed BK-induced HUVEC hyperpermeability and 0.4 mg/mL possessed the maximal inhibitory effect (87%, 70%, and 57% inhibition rate at 5, 15, and 30 min time point, respectively). Moreover, AEBO has presented remarkable IC50 = 0.24 mg/mL for anti-PLC activity, 0.36 mg/mL for anti-NO production, and 0.19 mg/mL for anti-cGMP production. For PKC inhibition, the IC50 (0.42 mg/mL) was slightly higher compared to others. Conclusion: This study provided supportive evidence for the previous study where AEBO exhibited anti-inflammatory activity against BK in vivo. The anti-inflammatory activity of AEBO may partly be associated with the reduction of endothelial hyperpermeability via the suppression of PLC-NO-cGMP signaling and PKC activity.