Lichens are a life form in which algae and fungi have a symbiotic relationship. A lichen has various biological activities, including anti-inflammatory and anti-proliferative activities. Inflammation is a response caused by various factors, such as infection by pathogens or tissue damage; excessive reactions can contribute to the etiology of chronic diseases, such as asthma, brain damage, and serious tissue damage. This study demonstrates the anti-inflammatory effect of ethyl acetate extract from Phlebia sp. on NF-κB and AP-1 pathways in the lipopolysaccharide-treated RAW 264.7 cell. Especially, Phlebia sp. extract inhibits the phosphorylation of AP-1 signaling (c-Fos and c-Jun) and its upstream MKK/MAPKs (MKK4, MKK7 and JNK), which induced a decrease in the production of the inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-1β (IL-1β), and granulocyte-macrophage colony-stimulating factor (GM-CSF) in downstream of AP-1 signaling. Furthermore, Phlebia sp. extract inhibited the production of final inflammatory effector molecules involved in AP-1 signaling, including nitric oxide (NO) and prostaglandin E2 (PGE2). Here, we report that Phlebia sp. extract has the potential to be developed as an anti-inflammatory agent.