Postmenopausal osteoporosis, mainly caused by osteoclast-induced bone resorption, has become a global public health burden. Natural compounds are emerging as potential therapeutics for postmenopausal osteoporosis. In vitro osteoclastogenesis assay was conducted to investigate the effect of Glycyrrhizic acid (Gly) on osteoclast differentiation without cytotoxity. We applied bone resorption pit assessment and F-actin immunofluoresence to explore the effect of Gly on osteoclasts function in vitro. RT-qPCR was used to evaluate the expression level of RANKL-induced osteoclast-specific gene. Western blotting was conducted for analyzing potential mechanisms of inhibitory influence of Gly on the formation and function of osteoclasts in vitro. Ovariectomized (OVX) mice model, micro-CT and histomorphometry was used to survey the potential effect of Gly on the resorption of bone in vivo. Gly inhibited osteoclast differentiation and function without significant cytotoxicity at a dose of no more than 8 mM in vitro. Gly attenuated mRNA expression of osteoclast-specific genes including NFATc1, c-fos, TRAP, CTR, cathepsin K, and V-ATPase d2 in vitro. Gly inhibited degradation of IκBα, phosphorylation of ERK and JNK without disturbing phosphorylation of p38 after treating osteoclasts in vitro. In OVX mice, Gly attenuates osteoclast formation and preserves bone mass and trabecular structure. Gly can effectively inhibit osteoclast maturation and bone resorption by suppressing NF-κB, ERK, and JNK pathway in vitro and exhibits an osteoprotective effect in OVX mice.