Pancreatic cancer is a highly aggressive disease with a very high mortality rate among all human cancers. The poor prognosis is in part due to intrinsic resistance to the apoptosis-inducing effects of radio- and chemotherapy. To find al ternative cell death pathways that can bypass the apoptotic resistance of pancreatic cancer cells, we examined the role of the novel anoikis effector Bit1 (Bcl-2 inhibitor of transcription) in the survival and apoptotic resistance of pancreatic cancer cells. Bit1 is a mitochondrial protein that induces a caspase-independent apoptosis upon its release into the cyto sol following loss of integrin-mediated attachment to extracellular matrix (anoikis). In this report, we observed that ec topic expression of Bit1 in the cytosol reduce d viability and induce d caspase-independent apoptosis in human pancreatic cancer cell lines, Miapaca-2 and PANC-1. While increased expression of mitochondrial Bit1 in these cells did not induce apoptosis under attached conditions, detachment significantly induced higher level of apoptosis in mitochondrial Bit1-transfected cells than in control transfected cells. Conversely, downregulation of endogenous Bit1 in PANC-1 cells further enhanced their anoikis resistance. Furthermore, exogenous expression of mitochondrial Bit1 in Miapaca-2 cells inhibited their anchorage-independent growth and enhanced their sensitivity to etoposide-mediated apoptosis. Mecha nistically, we found that the Bit1 apoptosis function is in part dependent on the groucho related Amino-terminal En hancer of Split (AES) expression and is abrogated by the transcriptional corepressor TLE1 protein. Consistent with our in vitro findings that Bit1 is an effector of apoptosis in pancreatic tumor cells, we fi nd that Bit1 is significantly down regulated in a fraction of advanced stages of human pancreatic carcinoma tissues. Taken together, these findings indi cate that the Bit1-apoptotic pathway can be targeted to trigger cell death in pancreatic cancer cells and implicate Bit1 as a novel therapeutic agent in attenuating pancreatic chemoresistance.