Extraocular muscles (EOMs) are a highly specialized type of tissue with a wide range of unique properties, including characteristic innervation, development, and structural proteins. Even though EOMs are frequently and prominently affected by thyroid-associated diseases, little is known about the direct effects of thyroid hormone on these muscles. To create a comprehensive profile of changes in gene expression levels in EOMs induced by thyroid hormone, hyperthyroid conditions were simulated by treating adult Sprague-Dawley rats with intraperitoneal injections of the thyroid hormone 3,3',5-triiodo-L-thyronine (T(3)); subsequently, microarray analysis was used to determine changes in mRNA levels in EOMs from T(3)-treated animals relative to untreated control animals. The expression of 468 transcripts was found to be significantly altered, with 466 of these transcripts downregulated in EOMs from T(3)-treated animals. The biological processes into which the affected genes could be grouped included cellular metabolism, transport, biosynthesis, protein localization, and cell homeostasis. Moreover, 15 distinct biochemical canonical pathways were represented among the genes with altered transcription levels. Strikingly, myostatin (Gdf8), a potent negative regulator of muscle growth, was found to be strongly downregulated in EOMs from T(3)-treated animals. Together, these findings suggest that pathological concentrations of thyroid hormone have a unique effect on gene expression in EOMs, which is likely to play a hitherto neglected role in thyroid-associated ophthalmopathies.