Osteoporosis is a chronic skeletal condition characterized by low bone mass and deteriorated microarchitecture of bone tissue, putting tens of millions of people at high risk of fractures. New therapeutic agents like i-body, a class of next-generation single-domain antibody, are needed to overcome some limitations of conventional treatments. An i-body is a human immunoglobulin scaffold with two long binding loops that mimic the shape and position of those found in shark antibodies the variable new antigen receptors (VNARs) of sharks. Its small size (~12kDa) and long binding loops provide access to drug targets which are undruggable by traditional monoclonal antibodies. We have successfully identified a human Receptor activator of nuclear-factor kappa Β (RANK) ligand (hRANKL) i-body, ADR3, which demonstrated high binding affinity to hRANKL with no adverse effect on the survival or proliferation of bone marrow-derived macrophages (BMMs). A differential scanning fluorimetry assay suggested that ADR3 was stable and able to tolerate a wide range of physical environments (temperature and pH). In vitro studies showed a dose-dependent inhibitory effect of ADR3 on osteoclast differentiation, podosome belt formation and bone resorption activity. Further investigation on mechanism of action of ADR3 revealed that it could block RANKL-mediated signalling pathways, supporting the in vitro functional observations. Collectively, these data indicate that RANKL antagonist ADR3, a next generation i-body attenuates osteoclast differentiation and bone resorption, representative of a novel therapeutic to protect bone loss such as osteoporosis and orthopaedic implant loosening.