Gemcitabine resistance will occur by time after the initial response in pancreatic cancer. Ginkgolide B (GB), a major terpene lactone component of Ginkgo biloba leaves, is a highly selective and competitive inhibitor for platelet-activating factor (PAF) receptor. In the present study, we evaluated the effect of GB on gemcitabine sensitivity in pancreatic cancer cell lines in vitro and in vivo. Cell viability assay, flow cytometry, dual luciferase reporter assay and tumor xenograft model were used to evaluate cell proliferation, apoptosis, nuclear factor kappa b (NF-кB) activity in vitro and tumor growth in vivo. Western blot, immunohistochemistry (IHC) and immunofluorescence were used to shown different protein expression levels. We found the half maximal inhibitory concentration (IC50) of gemcitabine was significantly downregulated by GB in a dose-dependent manner. Furthermore, GB could suppress cell proliferation, increase cell apoptosis and repress tumor growth when combined with gemcitabine, but had no effect when treated alone. Gemcitabine could upregulate PAFR and phosphorylated NF-кB/p65 expression, and increase NF-кB activity, but this was largely suppressed in combination with GB. GB could suppress PAFR expression in a dose-dependent manner. Knockout of PAFR significantly decreased phosphorylated NF-кB/p65 expression, inhibited NF-кB activity, increased gemcitabine sensitivity and cell apoptosis. Besides, GB had no influence on gemcitabine IC50 in IκBα-SR stably expressed BxPC-3 and CAPAN1. Our results suggested that GB could enhance gemcitabine sensitivity in pancreatic cancer cell lines by suppressing PAFR/NF-кB pathway. Thus GB may have therapeutic potential when used in combination with gemcitabine in pancreatic cancer.