Magnesium homeostasis is very important to normal functions of cell and plays a role in a number of diseases such as diabetes, hypertension, chronic respiratory disorder, osteoporosis and arrhythmia. MRS2 encode a magnesium transporter protein in Saccharomyces cerevisiae and deletion of MRS2 gene results in decrease in mitochondrial magnesium concentration, groupⅡ RNA splicing defect and growth defect on nonfermentable carbon source. To obtain more information of magnesium homeostasis in mitochondria, mTn-lacZ/LEU2 transposon library was transformed into mrs2 deletion mutant to screen for suppressor genes of MRS2. YMR166C, a member of mitochondrial carrier family, was identified as a suppressor gene of MRS2. Deletion of YMR166C gene can rescue the defects of mrs2 deletion mutant such as the decrease in mitochondrial magnesium concentration, groupⅡ RNA splicing defect and growth defect on nonfermentable carbon source. For the first time it was demonstrated that YMR166C is involved in mitochondrial magnesium homeostasis.