Pancreatic ductal adenocarcinoma (PDAC) has poor prognosis and high mortality rate. Inhibitor of differentiation-1 (ID-1) overexpression has already been reported to be associated with low survival, but the detailed roles of ID-1 in PDAC are unclear. As an ID-1 inhibitor, 2-methoxyestradiol (2-ME) has been shown effective in the antitumor therapies, but its effect on PDAC is unknown. In this study, ID-1 overexpression and knockdown stable SW1990 cells were used to examine proliferation, migration and invasion abilities. Effectiveness of 2-ME was estimated in vivo, as well as in vitro by survival analysis, magnetic resonance imaging and tissue analysis. PDAC patients were retrospectively reviewed to assess the ID-1 expression and prognosis. We found that ID-1 was closely associated with in vitro SW1990 cells proliferation, migration and invasion abilities, as well as the expression of HIF-1α, VEGF and MMP-9. 2-ME was shown to be effective on tumor suppression both in vivo and in vitro, perhaps mainly by ID-1 regulation. Moreover, the relevance between high ID-1 expression and poor prognosis was validated in PDAC patients. Our data collectively reveals the roles of ID-1 in PDAC malignancy, and suggests 2-ME treatment may be a potential alternative chemotherapeutic agent for PDAC patients.