Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignancies because of its broad resistance to chemotherapy. Numerous evidence indicates that integrinβ1 is upregulated in some human cancers, and it is correlated with resistance to various therapies. However, the role of integrinβ1 in chemotherapy is not clear in pancreatic cancer. The present study evaluates the potential of integrinβ1 to predict chemoresistance and prognosis in patients and to modulate resistance to gemcitabine in PDAC cells. Primary drug-resistance (DR) cancer cells were isolated, and DR cells from MiaPaCa-2 and AsPC-1 parent cell lines (PCL) were selected. Integrinβ1 expression was determined using immunohistochemistry (IHC), quantitative real-time PCR (qRT-PCR) and Western blotting. Changes in drug response after knockdown of integrinβ1 via RNA interference (RNAi) were evaluated using the viability of cancer cells as colon formation, proliferation using Western blot of Ki-67 and apoptosis using cleaved caspase-3 immunofluorescence. qRT-PCR and Western blot also detected variations in the activities of cdc42 and AKT after integrinβ1 suppression. Patient survival and relative factors were assessed using Kaplan-Meier and Cox regression analyses. Integrinβ1 expression was upregulated in PDAC, which was significantly associated with intrinsic and acquired gemcitabine resistance and worse outcomes. The downregulation of integrinβ1 attenuated PDAC chemoresistance, and this attenuation partially correlated with reduced Cdc42 and AKT activity, which are target molecules of integrinβ1 in some human cancers. These findings identified integrinβ1 as a special marker of drug resistance and a serious prognosis, and they furthermore support the use of integrinβ1 as a novel potential therapeutic target to overcome chemotherapy resistance. The results also suggest a possible drug-resistant signalling pathway of integrinβ1 in PDAC.