Our previous studies indicated that combination of Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and PPARγ ligand Troglitazone (TZD), can induce significant apoptosis in various TRAIL-resistant prostate and hepatocellular carcinoma (HCC) cells. These also suggested serine/threonine kinase AMP-activated protein kinase (AMPK) to be a mediator of TRAIL-TZD-induced apoptosis. To further validate AMPK's role in TRAIL sensitization, we determined the apoptotic potential of TRAIL in combination with the natural compound Berberine (BBR), the latter being a potent activator of AMPK. These demonstrated a significant reduction of cell viability and induction of apoptosis (increased cleavage of caspase 3, 8, 9) when treated with TRAIL-BBR combination. This apoptosis is attenuated in cells overexpressing AMPKα-dominant negative (DN) or following AMPKα knockdown, confirming involvement of AMPK. To identify potential downstream mediators involved, an apoptosis RT2 PCR array analysis was performed. These showed induction of several genes including TNFRSF10B (expresses DR5) and Harakiri following BBR treatment, which were further validated by qPCR analysis. Furthermore, knocking down DR5 expression significantly attenuated TRAIL-BBR-induced apoptosis, suggesting DR5 to be a mediator of this apoptosis. Our studies indicate that combination of TRAIL and AMPK activator BBR might be an effective means of ameliorating TRAIL-resistance involving DR5 in advanced cancer.