Botulinum neurotoxins (BoNT) are used in masticatory muscles for pain relief, unloading of the mandible, and cosmetic facial contouring. Treatment is often repeated every few months as function returns. This study assessed masticatory function and musculoskeletal structure after multiple BoNT treatment of the rabbit masseter. Female rabbits received 3 injections of BoNT (n = 13) or saline (n = 5) into one masseter muscle at intervals of 12 weeks. The contralateral side served as control. Periodic measurements of masticatory electromyography (EMG) and stimulated anterior bite force were made. After the final 12-week recovery interval, neuromuscular connection was investigated by stimulating the masseteric nerve to elicit an evoked EMG response. Mandibular specimens were collected for microCT analysis, and masseters were collected for histomorphometry and counts of replicating cells. Control and saline-injected muscles maintained consistent masticatory EMG and anterior bite force throughout the study. BoNT-injected masseters showed strong declines after each injection; during the 12-week recovery period, masticatory EMG and anterior bite force improved, although only electrical activity reached normal levels. Multiple injection resulted in persistently atrophied muscle fibers with fibrosis, and notable loss of bone from the mandibular body and condyle. The uninjected masseters of the BoNT group also showed evidence of mild toxin-related changes. Although muscle function is mostly regained after each injection, masseters receiving multiple doses of BoNT show extensive damage. In addition, mandibular bone density is decreased on the injected side. •Repeated botulinum treatment of the masseter reduced anterior bite force.•Fiber atrophy and fibrosis were prominent in the injected muscle.•Bone density in the mandibular body and condyle diminished with repeated injections.•After repeated treatment, side effects were seen in the contralateral masseter and condyle.