Skeletal muscle fatigue is a symptom of hyperthyroidic patients. The exact mechanism was not understood but smoothelin-like protein 1 (SMTNL1) was suspected to be the cause. Therefore, in this experiment, we investigated the effect of hyperthyroidism and overexpression of SMTNL1 protein on the C2C12 mouse myoblast cell line. To accomplish overexpression, we transfected the myoblasts with SMTNL1. We then studied the effect of hyperthyroidism by growing the transfected myoblasts under hyperthyroid conditions and comparing it to the control in a scratch assay to see the effect it would have on myoblast migration. We specifically wanted to check the effect of hyperthyroidism and SMTNL1 overexpression on the levels of phosphorylated and unphosphorylated MLC20 and MYPT1. We noticed that SMTNL1 overexpression and hyperthyroidism impeded cell migration in the scratch assay, SMTNL1 overexpression and hyperthyroidism have also increased phosphorylation of MYPT1. MYPT1 is deactivated when it’s phosphorylated and hence cannot dephosphorylate MLC20.