Osteosarcoma (OS) is one the most common primary malignancies of the bone in children and young adults with high metastasis. The use of non-toxic naturally derived compounds is one of present strategies in OS therapy to reduce secondary effects and chemo-resistance. Lactoferrin (LF), a transferrin protein derived from milk, currently appears to be an anticancer agent. However, its suppressive effects on OS have not been fully investigated. Therefore, we aimed to examine the molecular mechanism underlying the inhibitory effects of bovine LF (bLF) on OS. OS cell lines (NOS1, U2OS, MG63, and 143B) and an osteoblastic (ST2) were treated with bLF. Effects of bLF on OS-cell proliferation and migration were examined by proliferation and wound-healing assays. Expression levels of low-density-lipoprotein receptor-related protein 1 (LRP1) and cytokines including interleukin-1 beta (IL-1 beta), IL-6, and receptor activator of nuclear factor kappa-B ligand (RANKL) were measured using western blotting. Osteoclast formation was examined by co-culture of 1438, ST2, and bone marrow cells. We found that bLF down-regulated IL-1 beta, IL-6, and RANKL expression and suppressed phosphorylation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kappa B) p65 in 143B cells; bLF also drastically suppressed 143B-activated RANKL production in ST2 cells. This may have contributed to the reduction in the number of differentiated osteoclasts. Taken together, these data reveal that bLF down-regulates NF-kappa B to attenuate proliferation, migration, and bone resorption in OS and the OS-microenvironment. This study provides new findings and the precise underlying mechanisms of the inhibitory effects of bLF on OS. bLF can be a possible therapeutic agent for OS patients. (C) 2018 Elsevier Inc. All rights reserved.