A unilateral injection of botulinum toxin A (BTxA) in the calf induces paralysis and profound loss of ipsalateral trabecular bone within days. However, the cellular mechanism underlying acute muscle paralysis-induced bone loss (MPIBL) is poorly understood. We hypothesized that MPIBL arises via rapid and extensive osteoclastogenesis. We performed a series of in vivo experiments to explore this thesis. First, we observed elevated levels of the proosteoclastogenic cytokine receptor activator for nuclear factor-κB ligand (RANKL) within the proximal tibia metaphysis at 7 d after muscle paralysis (+113%, P<0.02). Accordingly, osteoclast numbers were increased 122% compared with the contralateral limb at 5 d after paralysis (P=0.04) and MPIBL was completely blocked by treatment with human recombinant osteoprotegerin (hrOPG). Further, conditional deletion of nuclear factor of activated T-cells c1 (NFATc1), the master regulator of osteoclastogenesis, completely inhibited trabecular bone loss (-2.2±11.9%, P<0.01). All experiments included negative control assessments of contralateral limbs and/or within-animal pre- and postintervention imaging. In summary, transient muscle paralysis induced acute RANKL-mediated osteoclastogenesis resulting in profound local bone resorption. Elucidation of the pathways that initiate osteoclastogenesis after paralysis may identify novel targets to inhibit bone loss and prevent fractures.