Previous studies have identified a positive correlation between the intake of n‑3 fatty acids and bone mineral density in postmenopausal women. The aim of the present study was to determine the effects of fish oil on bone metabolism and to investigate the underlying mechanism using ovariectomized rats. Ovariectomized or sham‑operated (sham) female rats were fed AIN‑76A‑based diets containing 5% corn or fish oil for 2 weeks. Fish oil was found to decrease the plasma levels of arachidonic and linoleic acids, but increased the levels of eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids. Fish oil reversed the increased activity and number of osteoclasts, and decreased calcium (Ca) and hydroxyproline (Hyp) content of the proximal tibia to sham values without affecting the activity or number of osteoblasts. In addition, fish oil suppressed increases in the mRNA and protein levels of macrophage colony‑stimulating factor (M‑CSF), PU.1, microphthalmia-associated transcription factor (MITF), receptor for activation of NFκB (RANK) and RANK ligand (RANKL) and serum levels of tumor necrosis factor α (TNFα), interleukin‑6 (IL-6) and prostaglandin E2 (PGE2). Fish oil was also found to suppress NFκB activation induced by ovariectomy. These results indicate that increases in plasma n‑3 fatty acid levels by fish oil led to the suppression of NFκB activation and subsequent downregulation of TNFα, followed by suppression of M‑CSF and RANKL. Dietary fish oil suppressed ovariectomy‑stimulated osteoclastogenesis by inhibiting the expression of M‑CSF, PU.1, MITF and RANK in the early stages of osteoclastogenesis, upstream of RANKL signaling.