Bone is a frequent target of lung cancer metastasis, which is associated with significant morbidity and a dismal prognosis. This study analyzed the soluble factors secreted by lung cancer cells, which are responsible for increasing osteoclast differentiation. Addition of recombinant human interleukin-8 (rhIL-8), present in large amounts in A549-conditioned medium (CM) and NCI-H460-CM, mimicked the inductive effect of A549-CM and NCI-H460-CM on osteoclastogenesis. In contrast, depletion of interleukin-8 (IL-8) from A549-CM and NCI-H460-CM decreased the osteoclastogenesis-inductive properties of A549-CM and NCI-H460-CM. Induction of osteoclast differentiation by lung cancer-derived-CM and rhIL-8 was associated with increased phospholipase D (PLD) activation, and the activations of protein kinase C (PKC) alpha/betaII, extracellular signal-regulated kinase (ERK) 1/2 and AKT/the mammalian target of rapamycin (mTOR). Blocking PLD by a specific inhibitor significantly decreased osteoclast formation by inhibiting PKCs activation and subsequently attenuating the phosphorylation of ERK1/2. PLD inhibitor also completely decreased AKT and mTOR phosphorylation, whereas phosphatidylinositol-3-kinase (PI3K) inhibitor only partially decreased mTOR phosphorylation, suggesting that mTOR activation by PLD is through both PI3K/AKT-dependent and PI3K/AKT-independent manner. In addition, blocking AKT and ERK1/2 by a specific inhibitor also suppressed lung cancer-derived-CM and rhIL-8-induced osteoclast differentiation. Moreover, treatment of peripheral blood mononuclear cells with sera from invasive lung cancer patients increased the formation of osteoclasts. Our study suggests that IL-8 or IL-8-mediated PLD/PKC/ERK1/2 or PLD/AKT signaling is an attractive therapeutic target for osteolytic bone metastases in lung cancer patients.