Despite progresses in diagnosis and treatment, pancreatic cancer continues to have the worst prognosis of all solid malignant tumors. Recent evidences suggest that the metastasis-promoting protein S100P stimulates pancreatic tumor proliferation, survival, invasion and metastasis progression through extracellular functions. Moreover, its expression is strongly correlated with poor prognosis in patients with several types of cancer although the entire molecular mechanism responsible for the diverse biological functions is not fully understood. We showed that extracellular S100P stimulates pancreatic carcinoma BxPC3 cell line by promoting cell proliferation. We also demonstrated that S100P induces, in this cell line, the phosphorylation of IκBα and the secretion of matrix metalloproteinase 9 (MMP-9). In addition, treatment with S100P protected cells from injuries induced by the cytotoxic agent Gemcitabine. On the basis of these results, we developed function-blocking anti-S100P monoclonal antibodies (mAbs) that abolished all of its in vitro activities. Furthermore, in vivo treatment with the candidate 2H8 antibody decreased tumor growth and liver metastasis formation in a subcutaneous and orthotopic BxPC3 tumor model. We conclude here that a therapeutic strategy blocking the extracellular activity of S100P by means of specific mAbs could be an attractive therapeutic approach as a single agent or in combination with target-directed or chemotherapeutic drugs to treat pancreatic cancer.