Excessive osteoclastogenesis and enhanced bone resorption are pathological hallmarks for bone diseases including osteolytic diseases, osteoporosis, and arthritis. Treatments targeting highly activated osteoclasts are regarded as promising therapies for osteoclast-related bone disorders. Cytochalasins are known as secondary metabolites of fungi and exhibit a variety of biological activities in cell biology and medicine. Cytochalasin Z11 (CytoZ11) was previously isolated from the Endothia gyrosa through solid substrate culture and showed therapeutic potential for leukaemia. However, the effects of CytoZ11 on osteoclasts currently remain unclear. Herein, CytoZ11 was found to be able to attenuate RANKL (receptor activator of nuclear factor-κB ligand)-induced osteoclastogenesis and bone resorptive activity dose-dependently. CytoZ11 could also inhibit mRNA expression of osteoclast-specific genes such as Ctr, Acp5, and Ctsk. Furthermore, CytoZ11 was demonstrated to suppress NFATc1 activation, which is due to the attenuation of two signaling pathways: c-Fos signaling and the NF-κB pathway. In summary, this study revealed that CytoZ11 may become a prospective drug for osteoclast-related disease by inhibiting osteoclast formation and function.