BACKGROUND Inflammation is a major cellular strain causing increased risk of osteo-degenerative diseases. Omega-3 fatty acids have been great source in suppressing inflammation. We investigated the effect of α-linolenic acid (ALA) on RANKL-stimulated osteoclast differentiation, LPS-induced and ovariectomized bone loss in mice models. MATERIAL AND METHODS The bone marrow macrophages (BMMs) were isolated from femurs of ICR mice, stimulated with RANKL, and treated with ALA (100, 200, 300 µM). Major analytical methods include histological analysis, osteoclasts viability assay, serum cytokines and chemokines ELISA, and gene expression by qPCR. RESULTS ALA intervention inhibited RANKL-induced osteoclasts proliferation and differentiation. ALA inhibited bone resorption activity as measured by materialization of F-actin ring structures as well. ALA suppressed the RANKL-induced osteoclast markers c-Fos, c-Jun and NFATc1 together with transcription factor proteins TRAP, OSCAR, cathepsin K and β3-integrin. ALA also suppressed the RANKL-stimulated phosphorylation of JNK, ERK, and AKT as well as NF-κB and BCL-2 proteins. ALA intervention (100 and 300 mg/kg) to LPS-challenged mice showed annulled morphometric changes induced by LPS by suppressing the levels of proinflammatory cytokines and chemokines. ALA (100 and 300 mg/kg) intervention to estrogen-deficiency induced bone loss mice (ovariectomized) showed reductions in TRAP+ osteoclasts count, CTX-I expression, levels of IL-1β, IL-2, IL-6, IL10, TNF-α and MCP-1 and iNOS and COX-2. CONCLUSIONS ALA suppresses RANKL-induced osteoclast differentiation and prevents inflammatory bone loss via downregulation of NF-κB-iNOS-COX-2 signaling. ALA is suggested to be a preventive herbal medicine against inflammatory bone disorders.