Arsenic is a well-known of human carcinogen and miR-301a is an oncogenic microRNA, which links to oncogenesis, however, little is understood about its contribution to arsenic-induced cellular transformation and tumorigenesis. Here, we investigated the role of miR-301a during arsenic-induced cellular transformation and tumor formation. miR-301a was found to be upregulated during arsenic-induced BEAS-2B transformation and the overexpression of miR-301a was dependent on IL-6/STAT3 signaling. Inhibition of miR-301a leads to reduction of cell proliferation, colony formation and cell migration. By using dual luciferase assay, SMAD4 was confirmed to be a direct target of miR-301a in BEAS-2B cells and upregulation of SMAD4 is involved the restraining cell growth and migration. In addition, reducing of miR-301a expression enhances doxorubicin-induced cellular apoptosis of transformed BEAS-2B through up-regulating SMAD4. Furthermore, we demonstrated that downregulation of miR-301a in BEAS-2B attenuates tumor growth in the xenograft model by targeting SMAD4. Of note, the level of miR-301a expression correlated inversely with SMAD4 expression in clinical specimens of human lung cancer. Our findings ascertain that miR-301a is an oncogenic miRNA, which targets SMAD4 to establish an essential mechanism for arsenic-induced carcinogenesis, IL-6/STAT3/miR-301a/SMAD4 signaling pathways.