The present study was designed to investigate the possible role of Mg²⁺ in suppression of phosphoenolpyruvate carboxy kinase (PEPCK) enzyme via inhibition of FOXO1gene expression in liver and we also examined whether Mg contributes to decrease blood glucose in muscle via inhibiting FOXO1 gene and protein expression. Fifty rats in five groups of experiment were considered as; non-diabetic control (NDC), Mg²⁺-treated non-diabetic control (Mg²⁺-NDC), chronic diabetic (CD), Mg²⁺-treated chronic diabetic (Mg²⁺-CD), and insulin-treated chronic diabetic (Ins-CD). Streptozotocin (STZ) was used for diabetes induction. The Mg²⁺-CD and Mg²⁺-NDC groups received 10 g/l of magnesium sulfate (MgSO₄) added to drinking water, and Ins-CD group received 2.5 U/kg of insulin. The blood glucose level and body weight were measured weekly. After 16 weeks, intraperitoneal glucose tolerance test (IPGTT) was done and blood samples were taken to determine the plasma levels of Mg and gastrocnemius muscle legs, and liver were isolated for both Forkhead transcription factor (FOXO1) and PEPCK enzyme genes and proteins expression. Administration of MgSO₄ improved IPGTT, lowered blood glucose levels and decreased FOXO1 and PEPCK genes and proteins expression in muscle and liver, while insulin just could decrease FOXO1 gene and protein expression in the muscle. These findings illustrated that MgSO₄ improved hyperglycemia via inhibition of FOXO1 gene and protein level in the muscle and liver, and it also decreased blood glucose level by prohibition of gluconeogenesis pathway in the liver. However, long time administration of insulin did not have any effect on liver.