Background: Use of botulinum toxin is becoming a popular way in treatment of some forms of strabismus.To evaluate the morphological changes in the orbital surface layer muscles of the rabbit eye following a single injection of botulinum toxin. Methods: 10 white Dutch rabbits weighing 2–3 kg received either 10 units (group 1; n=5) or 20 units (group 2; n=5) of botulinum toxin into the superior rectus muscle of right eye. The superior rectus muscle of the left eye in each rabbit was considered as control. The diameter of at least 20 muscle fibers of the orbital layer of the superior rectus muscle was measured with an ocular micrometer on histological sections after extirpation 2 and 10 days, and 4, 8 and 12 weeks after the injection. Results: Marked decrease in size of muscle fibers was observed, especially on 2 and 10 days post-injection. It was more sever in animals which received 20 units of the toxin. In both groups, between 4 and 12 weeks after injection, the size of some fibers returned back to normal; rarely, the fibers became hypertrophic while atrophic changes were still present. Hypertrophy of neuromuscular end-plate was seen accompanied with atrophic changes. Fibrosis around the muscle fibers was rarely seen. Conclusion: Injection of botulinum toxin into extraocular muscles causes atrophic changes which seems to be more severe with higher doses. The effect of botulinum toxin has been persisted up to 8 weeks.