The aim of this study was to investigate the chemical composition and anti-inflammatory effect of ethanolic extract of Brazilian green propolis (EEP-B) on LPS + IFN- γ or PMA stimulated J774A.1 macrophages. The identification and quantification of phenolic compounds in green propolis extract were performed using HPLC-DAD and UPLC-Q-TOF-MS methods. The cell viability was evaluated by MTT and LDH assays. The radical scavenging ability was determined using DPPH(•) and ABTS(•+). ROS and RNS generation was analyzed by chemiluminescence. NO concentration was detected by the Griess reaction. The release of various cytokines by activated J774A.1 cells was measured in the culture supernatants using a multiplex bead array system based on xMAP technology. Artepillin C, kaempferide, and their derivatives were the main phenolics found in green propolis. At the tested concentrations, the EEP-B did not decrease the cell viability and did not cause the cytotoxicity. EEP-B exerted strong antioxidant activity and significantly inhibited the production of ROS, RNS, NO, cytokine IL-1 α , IL-1 β , IL-4, IL-6, IL-12p40, IL-13, TNF- α , G-CSF, GM-CSF, MCP-1, MIP-1 α , MIP-1 β , and RANTES in stimulated J774A.1 macrophages. Our findings provide new insights for understanding the anti-inflammatory mechanism of action of Brazilian green propolis extract and support its application in complementary and alternative medicine.