Phospholipase C (PLC) and protein kinase C (PKC) are known for their vital role in regulating endothelial permeability. PKC induces endothelial permeability through disassembly of adherences junctions and increased actin polymerization, whereas PLC activation triggers production of downstream signaling molecules, such as intracellular calcium, nitric oxide and cyclic guanosine monophosphate (cGMP). Previous studies showed that Bixa orellana leaves extract attenuated acute inflammation that caused by snake venom [1] and bradykinin [2]. Therefore, the aim of this study was to investigate the effect of aqueous extract of B. orellana leaves (AEBO) on PLC and PKC activities in bradykinin-induced endothelial hyperpermeability. Bradykinin-induced hyperpermeability in human umbilical veins endothelial (HUVEC) was successfully suppressed by AEBO at 0.1 – 0.4 mg/ml after 12 h pre-treatment. AEBO at 0.4 mg/ml exerted maximum inhibition with a rate of 86.7%, 70.5% and 57.5%, at 5, 15 and 30 min, respectively. Inositol phosphate production, which represent PLC activity was significantly suppressed by AEBO at 0.2 mg/ml (to 51.9 ± 0.2 nM) and 0.4 mg/ml (to 89.4 ± 1.4 nM), but notat 0.1 mg/ml, compared to untreated control. Similar inhibitory effect on PKC activity was also observed in AEBO-treated group at 0.4 mg/ml, with a maximum rate of inhibition of 44.5%. However, there was no alteration on PKC activity detected in AEBO-treated group at 0.1 mg/ml. In conclusion, AEBO possesses anti-endothelial hyperpermeability effect against bradykinin through suppression of PLC and PKC activities.