Background: The high mobility group A1 (HMG A1) proteins are overexpressed in pancreatic cancers. They are architectural nuclear proteins, which regulate gene expression. We have previously shown that carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) is a molecular determinant of chemosensitivity in pancreatic adenocarcinoma. In this study, we hypothesize that HMG A1 is a nuclear regulator of CEACAM6 and hence, its silencing will promote chemosensitivity in pancreatic adenocarcinoma. Methods: We studied highly malignant pancreatic adenocarcinoma cell lines (BxPC3, MiaPaCa2 and PANC1). Four short-hairpin RNA (shHMGA1) expression vectors targeting different regions of HMG A1 were assessed for the degree of silencing of HMGA1 expression. Construct with greatest degree of silencing of HMGA1 was selected for generation of lentiviral particles. Stable transfectants were developed after lentiviral transduction. Full-length cDNA of HMGA1 was cloned into pIRES-puro vector and stably transfected into MiaPaCa2 cells with inherent underexpression of HMGA1 (pIRES-HMGA1.1 and 1.2). Nuclear expression of HMGA1 and cellular expression of CEACAM6 were confirmed using Western analysis. Chemosensitivity to gemcitabine was determined by IC50 analysis. Apoptosis was measured by caspase 3 activation using cleaved-caspase 3 ELISA assay. Akt kinase assay was performed based on GSK-3 phosphorylation. Results: Lentivirally-transduced RNA interference resulted in 90% silencing of HMG A1 expression in all cell lines. Silencing of HMGA1 resulted in a significant reduction in the CEACAM6 expression, and forced overexpression of HMGA1 elevated the expression of CEACAM6 (p<0.05 based on densitometry). Silencing of HMGA1 resulted in significant reduction in Akt kinase activity. Silencing of HMGA1 enhanced chemosensitivity to gemcitabine with two- to four-fold reduction in IC50 (all cell lines, p<0.05 vs. controls i.e. lentivirus carrying scrambled RNAi sequence). Lentivirus-mediated silencing of HMGA1 promoted the activation of caspase 3, a central mediator of apoptosis, on exposure to gemcitabine (p<0.05 versus controls). On the contrary, pIRES-HMGA1.1 and 1.2 MiaPaCa2 clones with forced overexpression of HMGA1 showed a decrease in chemosensitivity to gemcitabine, with significant two-fold increase in IC50 (p<0.05 vs. empty pIRES vector). Conclusions: HMG A1 is a molecular determinant of gemcitabine chemosensitivity in pancreatic adenocarcinoma, and this may in part be mediated by its regulatory role on the cellular expression of CEACAM6. Lentivirus-mediated RNA interference of HMGA1 may represent a feasible therapeutic option for pancreatic cancer.