Abstract: Background and Objective: The clinical use of chemotherapeutic drugs is normally restricted by severe side effects and drug resistances. Brucine is a natural alkaloid derived from the seeds of Strychnos nux-vomica Linn., which has been reported to possess antitumor activity. This study was aimed to investigate the effects of brucine on 5-fluorouracil (5-FU)-induced cell death in human liver carcinoma HepG2 cells. Materials and Methods: Cells were treated with 5-FU and brucine alone or in combination and cell viability was measured using MTT method. Apoptotic effects were analyzed by using acridine orange/ethidium bromide (AO/EB) staining and annexin V/PI staining assays. The mRNA expression of Fas/FasL pathway members including Fas, Fas ligand (FasL) and Fas-associated protein with the death domain (FADD) was detected by real-time PCR. Western blot analysis was further conducted to determine the expression of Fas and FasL proteins. Results: Combination treatment obviously decreased the viability of HepG2 cells compared to 5-FU treatment alone. Exposure of cells to 5-FU and brucine in combination lead to increased apoptosis as evidenced by staining with AO/EB and annexin V/PI. Real-time PCR assay showed that 5-FU in combination with brucine markedly enhanced the mRNA levels of Fas, FasL and FADD. In addition, the upregulation of Fas and FasL proteins expression was also confirmed by Western blot analysis. Conclusion: These findings suggested that brucine could enhance 5-FU-induced the cytotoxicity and apoptosis through Fas/FasL signaling pathway in HepG2 cells. Administration of 5-FU in combination with brucine may be an effective and feasible therapy for human liver carcinoma.