Coptis chinensis Franch (CCF) has been widely used by Chinese old herbalist doctor to treat internal and external diseases including malignant sore and cancer. Berberine (BBR) is a major bioactive compound in CCF and may exert anti-tumor and anti-inflammatory effects like CCF. However, the prevention effect of berberine against lung cancer and its relevance of anti-inflammation property to cancer-preventing effect are still obscure. Protein arginine deaminase 4 (PAD4) played an important role in macrophage related inflammatory response, the purpose of this study was to identify whether berberine can prevent lung cancer and explore its effect on PADI4-related macrophage function. In vitro, PADI4 overexpression affects cell-activated state in macrophages. PADI4 overexpressed macrophages promote epithelial-mesenchymal transition (EMT) of A549 lung cancer cells and inhibit cell apoptosis. Berberine at the experiment dose had no effect on cell viability of U937-derived macrophages, but could significantly inhibit PADI4 expression to reverse the macrophage-activated state and the lung cancer -promoting effect of PADI4-overexpressed macrophages. Unlike GSK484, berberine had a little effect on the PADI4 citrullination activity at the experimental doses, its IC50 for PADI4 inhibition is 45.07 μM (44.03-46.12 μM). In the mouse lung carcinogenetic model, PADI4 expression was directly related to the number of lung nodules. Berberine had the similar role to GSK484 in reducing the number of lung tumor nodules with the improved lung pathology in a dose-dependent manner and significantly inhibited PADI4 expression. Further, we found that PADI4 overexpression could inhibit IRF5 expression, up-regulate CD163 and CD206 and down-regulate CD86 in macrophages, which could be reversed by berberine. Our results suggest that berberine may regulate PADI4-related macrophage function to prevent lung cancer.