The discovering of an osteoclast (OC) coupling active agent, capable of suppressing OC-mediated bone resorption while concurrently stimulating osteoblast (OB)-mediated bone formation, presents a promising strategy to overcome limitations associated with existing antiresorptive agents. However, there is a lack of research on active OC coupling agents.This study aims to investigate the potential of Jiangu Formula (JGF) in inhibiting OCs while maintaining the OCOB coupling function.The anti-osteoporosis efficacy of JGF was evaluated in osteoporosis models induced by ovariectomy in C57BL/6 mouse and SD rats. The effect of JGF on OCs was evaluated by detecting its capacity to inhibit OC differentiation and bone resorption in an in vitro osteoclastogenesis model induced by RANKL. The OCOB coupling activity of JGF was evaluated by measuring the secretion levels of OC-derived coupling factors, OB differentiation activity of MC3T3-E1 interfered with conditioned medium, and the effect of JGF on OC inhibition and OB differentiation in a C3H10T1/2-RAW264.7 co-culture system. The mechanism of JGF was studied by network pharmacology and validated using western blot, immunofluorescence (IF), and ELISA. Following that, the active ingredients of JGF were explored through a chemotype-assembly approach, activity evaluation, and LC-MS/MS analysis.JGF inhibited bone resorption in murine osteoporosis without compromising the OCOB coupling effect on bone formation. In vitro assays showed that JGF preserved the coupling effect of OC on OB differentiation by maintaining the secretion of OC-derived coupling factors. Network analysis predicted STAT3 as a key regulation point for JGF to exert anti-osteoporosis effect. Further validation assays confirmed that JGF upregulated p-STAT3(Ser727) and its regulatory factors IL-2 in RANKL-induced RAW264.7 cells. Moreover, 23 components in JGF with anti-OC activity identified by chemotype-assembly approach and verification experiments. Notably, six compounds, including ophiopogonin D, ginsenoside Re, ginsenoside Rf, ginsenoside Rg3, ginsenoside Ro, and ononin were identified as OC-coupling compounds.This study first reported JGF as an agent that suppresses bone loss without affecting bone formation. The potential coupling mechanism of JGF involves the upregulation of STAT3 by its regulators IL-2. Additionally, the chemotype-assembly approach elucidated the activity compounds present in JGF, offering a novel strategy for developing an anti-resorption agent that preserves bone formation.Copyright © 2024 Elsevier GmbH. All rights reserved.