BACKGROUND: We previously reported Brucea javanica leaf extract (BJLE) induced apoptosis in human oral squamous cell carcinoma (HSC2) cells by attenuation of mitochondrial membrane permeability. However, further underlying mechanism is not known yet. Therefore, we conducted a study to investigate activation of Caspases related to attenuation of mitochondrial membrane permeability in BJLE-treated human oral squamous cell carcinoma.METHODS: B. javanica leaves were collected, identified, minced, dried, extracted with distilled ethanol at room temperature for 24 hours, filtered and evaporated. Resulted BJLE was stored at 4°C. HSC-2 and HSC-3 cells were fasted for 12 hours and treated with BJLE in various concentrations for 24 hours. Treated HSC-2 and HSC-3 cells were lysed and subjected to western blot, to detect cleaved-Caspase-9, cleaved-Caspase-3 and β-actin. All visualized bands were captured and quantified.RESULTS: Low numbers and morphological alterations of adherent HSC-2 and HSC-3 cells were observed in the group of cells treated with 500, 100 and 10 μg/mL BJLE. Numbers of adherent HSC-2 and HSC-3 cells treated with BJLE were shown decreased along with the increase of BJLE concentrations. Meanwhile, numbers of floating HSC-2 and HSC-3 cells were increased. Bands of cleaved-Caspase-9 and cleaved-Caspase-3 were observed in HSC-2 and HSC-3 cells treated with 500 and 100 μg/mL BJLE. Higher-density bands of cleaved-Caspase-9 and cleaved-Caspase-3 were observed in HSC-2 and HSC-3 cells treated with 500 μg/mL BJLE than 100 μg/mL BJLE. CONCLUSION: BJLE could induce apoptosis by activation of Caspase 9 and Caspase 3 of mitochondrial apoptotic pathway in human oral squamous cell carcinoma.