Abstract Background: M2 tumor-associated macrophages can promote pancreatic cancer progression. Under ROS, cancer cells derived IL-4 can skew M2 with uncertain mechanism. In this study, we tried to elucidate the mechanism and design gene therapy to treat pancreatic cancer by inhibiting M2 macrophages. Methods: We used CRISPR/Cas9 to knock out CYBB that encodes NOX2 in macrophages and adopted CYBB-/- B6 mice. Co-culture assay and syngeneic orthotopic pancreatic cancer mice models were used to study the mechanism in skewing M2 and tumor growth. The decoy oligodeoxynucleotides were designed to inhibit M2 formation and control the tumor growth. Results: Co-culture with pancreatic cancer cells induced M2 polarization at 72 h, while CYBB knockout prevented it. In the syngeneic orthotopic model, M2 macrophages count was decreased and tumor formation was inhibited in Cybb-/- mice. Under secretion of ROS in activated macrophages, cancer cell derived IL-4 can increase CD204 expression on macrophages. Chromatin immunoprecipitation study showed a binding site of STAT6 and HDAC2 in the promotor region of Cybb gene. Using epigenetic inhibitors or knocking down STAT6 and HDAC2 could block the M2 skewing with reducing the IL-10 expression and keep on M1 macrophages with increasing the IL-12 expression. Gene therapy using STAT6-CYBB decoy oligodeoxynucleotides to interrupt STAT6/HDAC2 binding to CYBB promoter could inhibit M2 formation and reduce tumor growth significantly.Conclusion: Gene therapy using STAT6-CYBB decoy ODN can inhibit M2 formation to treat pancreatic cancer.