The exact mechanisms underlying chemotherapy insensitivity in pancreatic cancer remain largely unclear. The dynamic cross talk between tumors and their microenvironment is an important determinant of cancer chemosensitivity. However, whether additional signals provided during the intravascular transit of tumor cells affect the sensitivity of pancreatic cancer cells to chemotherapy is unknown. We have found that activated platelet–cancer cell interactions are sufficient to prime cisplatin (CDDP) insensitivity in pancreatic cancer cells. Activated platelet-derived TGF-β1 rather than direct platelet–tumor cell contacts stimulates PI3K/Akt and MEK/Erk signaling in pancreatic cancer cells, resulting in reduction of CDDP sensitivity in these cells. Therefore, the platelet–tumor cell interactions and the relevant signaling pathways that prime CDDP insensitivity may be potential targets for adjuvant chemotherapy for pancreatic cancer.