Hypothyroidism is associated with muscle weakness and slowed movements. Hormone-deficient muscles do show changes in contractile proteins and sarcoplasmic calcium pumps, however effects on the components of the neuromuscular junction are less clear. In this study, we examined cholinesterase activity in adult fast-twitch muscle from hypothyroid and control rats. Male Holtzman rats underwent thyroidectomy and their age-matched euthyroid controls were simultaneously subjected to sham operation. Thirty days post-operative, animals were sacrificed for anterior tibialis muscles harvest. Muscle cholinesterase isoform activity was measured and compared between experimental treatment groups. Butyrylcholinesterase activities and peak I and III acetylcholinesterase (AchE) activities were similar in euthyroid and hypothyroid groups. However, hypothyroid muscles exhibited half the peak II AchE isoform (G4) activity, as compared to control muscles. Hypothyroidism specifically affects AchE isoform expression in rodent fast-twitch muscle.