Pancreatic cancer is characterized as inflammatory malignancy with a dismal prognosis. There is abundant intratumoral infiltration of macrophages, and most of these tumor associated macrophages (TAM) are induced to be M2 phenotype. The M2 polarized TAM has been demonstrated to promote progression and induce chemo-resistance of pancreatic cancer. Interleukin (IL)-27 is a novel member of IL-12 cytokine family and its roles in regulation of phenotypes and functions of TAM remain largely unknown. In this study, we demonstrated IL-27 significantly inhibited the M2 macrophages polarization and dampened the proliferation, migration and metastasis of pancreatic cancer cells and as well enhanced the efficacy of gemcitabine. IL-27 could be potential to improve the treatment of pancreatic cancer by targeting M2 polarized TAMs.