The role of pancreatic stellate cells (PSCs) has been established in many studies. However, the potential mechanism for the chemoresistance induced by PSCs has not been fully elucidated. In the present study, human pancreatic cancer cell lines were directly or indirectly co-cultured with PSCs. The inhibition rate and IC50 values were assessed to determine the ability of chemoresistance. RT-PCR and western blot analysis were used to evaluate Hes 1 and Jagged 1 expression before and after co-culture with PSCs. To determine the relationship between Hes 1 expression and survival in pancreatic cancer patients, Kaplan-Meier survival analysis was performed. PSCs promoted the expression of Hes 1 in both PANC-1 and BxPC-3 cell lines and induced chemoresistance to gemcitabine. A Notch signaling pathway inhibitor (L1790) and Hes 1 siRNA reversed the chemoresistance induced by PSCs. In 72 resected pancreatic cancer patients, high Hes 1 expression was observed in 34 patients with shorter overall and progression-free survival times. In conclusion, Hes 1 is essential for chemoresistance induced by PSCs and is associated with poor prognosis in pancreatic cancer patients. Therapy targeting the Notch signaling pathway may reverse chemoresistance and improve survival in patients with pancreatic cancer.