Heat Shock Factor 1 (HSF1) is a transcription factor for multiple cell survival proteins including HSP70. We hypothesize that HSF1 overexpression is vital for the survival of pancreatobiliary tumors and that inhibiting its expression induces apoptosis in pancreatiobiliary tumor cells. Methods: HSF1 expression was reduced in pancreatic (MiaPaCa-2 & S2013) and cholangiocarcinoma (KMCH, KMBC) cell lines by treatment with HSF1 siRNA. Two unique sequences of HSF1 siRNA were used to rule out any off-target effects of siRNA. Cell viability (MTT assay) and apoptosis (annexin V staining, caspase 3 and 9) were measured. Results: Inhibition of HSF1 expression by HSF1siRNA markedly reduced the viability of all the cancer cell lines at 96 h. Viability (% of control) expressed as mean ± SEM: MiaPaCa-2: 37.2 ± 3.2, KMCH 41.3 ± 7.61. Similar results were observed for the other two cell lines. Inhibition of HSF1 expression also led to increased annexin V staining in all the cell lines studied. Further, inhibition of HSF1 expression also led to caspase-3 activation in all the cancer cell line studied. Caspase 3 (% of control) expressed as mean ± SEM: MiaPaCa-2: 497.4 ± 24.3; KMCH: 465.2 ± 22.68. Similar results were observed when the HSF1 expression was inhibited by the other HSF1siRNA sequence. Conclusion: Silencing HSF1 expression activates caspase-dependent apoptotic cell death in pancreatobiliary cancer cells. HSF1 holds a great promise as a candidate for drug development.