Increased microRNA-10b (miR-10b) expression in the cancer cells in pancreatic ductal adenocarcinoma (PDAC) is a marker of disease aggressiveness. In the present study, we determined that plasma miR-10b levels are significantly increased in PDAC patients by comparison with normal controls. By gene profiling, we identified potential targets downregulated by miR-10b, including Tat-interacting protein 30 (TIP30). Immunoblotting and luciferase reporter assays confirmed that TIP30 was a direct miR-10b target. Downregulation of TIP30 by miR-10b or siRNA-mediated silencing of TIP30 enhanced epidermal growth factor (EGF)-dependent invasion. The actions of miR-10b were abrogated by expressing a modified TIP30 cDNA resistant to miR-10b. EGF-induced EGF receptor (EGFR) tyrosine phosphorylation and extracellular signal-regulated kinase phosphorylation were enhanced by miR-10b, and these effects were mimicked by TIP30 silencing. The actions of EGF in the presence of miR-10b were blocked by EGFR kinase inhibition with erlotinib and by dual inhibition of PI3K (phosphatidylinositol 3'-kinase) and MEK. Moreover, miR-10b, EGF and transforming growth factor-beta (TGF-β) combined to markedly increase cell invasion, and this effect was blocked by the combination of erlotinib and SB505124, a type I TGF-β receptor inhibitor. miR-10b also enhanced the stimulatory effects of EGF and TGF-β on cell migration and epithelial-mesenchymal transition (EMT) and decreased the expression of RAP2A, EPHB2, KLF4 and NF1. Moreover, miR-10b overexpression accelerated pancreatic cancer cell (PCC) proliferation and tumor growth in an orthotopic model. Thus, plasma miR-10b levels may serve as a diagnostic marker in PDAC, whereas intra-tumoral miR-10b promotes PCC proliferation and invasion by suppressing TIP30, which enhances EGFR signaling, facilitates EGF-TGF-β cross-talk and enhances the expression of EMT-promoting genes, whereas decreasing the expression of several metastasis-suppressing genes. Therefore, therapeutic targeting of miR-10b in PDAC may interrupt growth-promoting deleterious EGF-TGF-β interactions and antagonize the metastatic process at various levels.