Bone matrix homeostasis associated diseases such as osteoporosis and erosive arthritis were caused by the imbalance of osteoclast-mediated bone-resorption and osteoblast-mediated bone-formation. Suppressing the fusion and differentiation of osteoclast from osteoclast precursors are an essential way to maintain the dynamic balance of resorption and formation. Recently, some natural products were discovered to inhibit osteoclast formation and function for potential treatment of osteoporosis. Vanillin was previously reported to have anti-tumor and anti-oxidant activities; however, its effect on bone health has not been elucidated. In this study, we found that the inhibitory effect of vanillin on RANKL-induced multinucleated osteoclast formation and bone resorption (concentration of 0.25 mM–2.5 mM). Morphologically, the number of mature osteoclasts was decreased after treating with vanillin in a dose-dependent manner, which was evaluated by TRAP staining and FAK staining. Vanillin could significantly inhibit bone resorption and promote the early apoptosis rate during RANKL-induced osteoclastogenesis. Furthermore, vanillin could activate the mitochondrial-dependent apoptosis via inducing the expression of cytochrome c, cleaved caspease-3, BAX and Apaf-1 both on mRNA and protein level. Otherwise, the expression of Bcl-2 was inhibited. Thereby, these data provide the clue that vanillin could be a candidate to treat bone matrix metabolic diseases.