Objective: Calcium homeostasis plays an important role in many physiological processes, including muscle contraction, mitochondrial metabolism, and decision of cell fate. Recently it has been reported alterations in calcium homeostasis induces insulin resistance and diabetes. However, the relationship between calcium regulation and free fatty acid-insulin resistance in skeletal muscles has not been well studied. In this study, we investigated the relationships and molecular mechanisms between calcium-overload and insulin resistance in human skeletal muscle cells. Methods: We used differentiated human skeletal muscle cells and treated free fatty acid for inducing insulin resistance. Intracellular calcium was measured by Fluo-3/AM staining. Metabolism-related genes and proteins were analyzed by immunoblotting and qPCR. To investigate the effects of calcium metabolism on insulin sensitivity, muscle cells were treated with calcium supplementation reagents, which was achieved by palmitate, tapsigargin, and A23187 treatment, or calcium reduction reagent, which occurred by Bapta-AM administration. Results: Free fatty acid increases intracellular calcium concentration and at the same time induces insulin resistance in muscle cells. The calcium chelator, Bapta-AM, dramatically inhibited free fatty acid-induced insulin resistance, but calcium donors, tapsigargin and A23187 treatment, evoked insulin resistance. Bapta-AM dramatically inhibited free fatty acid-induced intracellular calcium flux, but calcium donors, tapsigargin or A23187, evoked intracellular calcium flux. Bapta-AM ameliorated free fatty acid-induced insulin resistance through improved insulin signaling such as AKT and GSK in human skeletal muscle cells. Glucose up take was also significantly recovered by Bapta-AM. The calcium chelator ameliorated activation of inflammatory signals such as JNK, p38, ER-stress, mitochondrial dysfunctions, and iron overload against free fatty acid. Conclusion: In this study, we investigated that fatty acid evoked intracellular calcium abnormalities and causes insulin resistance. Reduction of intracellular calcium flux with chelating agent significantly decreased insulin resistance. Therefore, calcium homeostasis in human skeletal muscle cells is crucial to controlling metabolic diseases such as insulin resistance and diabetes.