Purpose: To determine the effect of hyperthyroidism on both myofiber number and myosin heavy-chain isoform composition within the palpebral orbicularis oculi muscle in rabbits. Methods: Four New Zealand White rabbits were made hyperthyroid by injection of 3,3,3′-triiodothyroinine intraperitoneally every other day for 1 month. Four rabbits were used as control animals. After 1 month the rabbits were euthanized, and the eyelids were excised and sectioned in a cryostat. The sections were immunostained to determine the presence of fast, slow, and neonatal myosin heavy-chain isoforms. To determine alterations in myofiber number, differential counts of myofiber number and the cross-sectional areas of the muscle fibers were performed with the use of computerized morphometry. Results: The orbicularis oculi muscle in the palpebral portion of the eyelids from hyperthyroid rabbits had significantly fewer myofibers compared with control eyelids, predominantly as the result of a loss of myofibers in the preseptal region. The remaining fibers showed continued expression of fast myosin but upregulated coexpression of slow myosin isoform. Conclusions: Hyperthyroidism led to reduced orbicularis oculi muscle in the rabbit model and an alteration in the myosin heavy-chain isoform composition. This finding may help explain the clinical finding of eyelid retraction in patients with Graves orbitopathy.