beta-lapachone (beta-L) is a substrate of reduced nicotinamide adenine dinucleotide (NADH): quinone oxidoreductase 1 (NQO1). NQO1 reduces quinones to hydroquinones using NADH as an electron donor and consequently increases the intracellular NAD+-/NADH ratio. The activation of NQO1 by beta-L has beneficial effects on several metabolic syndromes, such as obesity, hypertension, and renal injury. However, the effect of beta-L on bone metabolism remains unclear. Here, we show that beta-L might be a potent inhibitor of receptor activator of nuclear factor-kappa B ligand (RANKL)-induced osteoclastogenesis. beta-L inhibited osteoclast formation in a dose-dependent manner and also reduced the expression of osteoclast differentiation marker genes, such as tartrate-resistant acid phosphatase (Acp5 or TRAP), cathepsin K (CtsK), the d2 isoform of vacuolar ATPase V0 domain (Atp6v0d2), osteoclast-associated receptor (Oscar), and dendritic cell-specific transmembrane protein (Dc-stamp). beta-L treatment of RANKL-induced osteoclastogenesis significantly increased the cellular NAD+/NADH ratio and resulted in the activation of 5' AMP-activated protein kinase (AMPK), a negative regulator of osteoclast differentiation. In addition, beta-L treatment led to significant suppression of the expression of peroxisome proliferator-activated receptor gamma (PPAR gamma) and peroxisome proliferator-activated receptor gamma coactivator 1 beta (PGC1 beta), which can stimulate osteoclastogenesis. beta-L treatment downregulated c-Fos and nuclear factor of activated T-cells 1 (NFATc1), which are master transcription factors for osteoclastogenesis. Taken together, the results demonstrated that beta-L inhibits RANKL-induced osteoclastogenesis and could be considered a potent inhibitor of RANKL-mediated bone diseases, such as postmenopausal osteoporosis, rheumatoid arthritis, and periodontitis. (C) 2016 Elsevier Inc. All rights reserved.