Pancreatic cancer is one of the malignant diseases with the worst prognosis. Resistance to chemotherapy is a major difficulty in treating the disease. We analyzed plasma samples from a genetically engineered mouse model of pancreatic cancer and found soluble vascular cell adhesion molecule-1 (sVCAM-1) increases in response to gemcitabine treatment. VCAM-1 was expressed and secreted by murine and human pancreatic cancer cells. Subcutaneous allograft tumors with overexpression or knock-down of VCAM-1, as well as VCAM-1-blocking treatment in the spontaneous mouse model of pancreatic cancer, revealed that sVCAM-1 promotes tumor growth and resistance to gemcitabine treatment in vivo but not in vitro. By analyzing allograft tumors and co-culture experiments, we found macrophages were attracted by sVCAM-1 to the tumor microenvironment and facilitated resistance to gemcitabine in tumor cells. In a clinical setting, we found that the change of sVCAM-1 in the plasma of patients with advanced pancreatic cancer was an independent prognostic factor for gemcitabine treatment. Collectively, gemcitabine treatment increases the release of sVCAM-1 from pancreatic cancer cells, which attracts macrophages into the tumor, thereby promoting the resistance to gemcitabine treatment. sVCAM-1 may be a potent clinical biomarker and a potential target for the therapy in pancreatic cancer.