NF‐κB is a transcriptional activator of many genes, including some that lead to muscle atrophy and bone resorption—significant concerns for astronauts. NF‐κB activation is inhibited by eicosapentaenoic acid (EPA), but the influence of this omega‐3 fatty acid on the effects of weightlessness are unknown. We report here cellular, ground analogue, and spaceflight findings. We investigated the effects of EPA on differentiation of RAW264.7 monocyte/macrophage cells induced by receptor activator of NF‐κB ligand (RANKL) and on activation of NF‐κB by tumor necrosis factor α (TNF‐α) or exposure to modeled weightlessness. EPA (50 µM for 24 hours) inhibited RANKL‐induced differentiation and decreased activation of NF‐κB induced by 0.2 µg/mL of TNF‐α for 30 minutes or by modeled weightlessness for 24 hours (p < .05). In human studies, we evaluated whether NF‐κB activation was altered after short‐duration spaceflight and determined the relationship between intake of omega‐3 fatty acids and markers of bone resorption during bed rest and the relationship between fish intake and bone mineral density after long‐duration spaceflight. NF‐κB was elevated in crew members after short‐duration spaceflight, and higher consumption of fish (a rich source of omega‐3 fatty acids) was associated with reduced loss of bone mineral density after flight (p < .05). Also supporting the cell study findings, a higher intake of omega‐3 fatty acids was associated with less N‐telopeptide excretion during bed rest (Pearson r = –0.62, p < .05). Together these data provide mechanistic cellular and preliminary human evidence of the potential for EPA to counteract bone loss associated with spaceflight. © 2010 American Society for Bone and Mineral Research