Bone receives mechanical stimulation from two primary sources, muscle contractions and external gravitational loading; but the relative contribution of each source to skeletal health is not fully understood. Understanding the most effective loading for maintaining bone health has important clinical implications for prescribing physical activity for the treatment or prevention of osteoporosis. Therefore, we investigated the relative effects of muscle paralysis and reduced gravitational loading on changes in muscle mass, bone mineral density, and microarchitecture. Adult female C57Bl/6J mice (n = 10/group) underwent one of the following: unilateral botulinum toxin (BTX) injection of the hind limb, hind limb unloading (HLU), both unilateral BTX injection and HLU, or no intervention. BTX and HLU each led to significant muscle and bone loss. The effect of BTX was diminished when combined with HLU, though generally the leg that received the combined intervention (HLU+BTX) had the most detrimental changes in bone and muscle. We found an indirect effect of BTX affecting the uninjected (contralateral) leg that led to significant decreases in bone mineral density and deficits in muscle mass and bone architecture relative to the untreated controls; the magnitude of this indirect BTX effect was comparable to the direct effect of BTX treatment and HLU. Thus, while it was difficult to definitively conclude whether muscle force or external gravitational loading contributes more to bone maintenance, it appears that BTX-induced muscle paralysis is more detrimental to muscle and bone than HLU.