Duchenne muscular dystrophy (DMD) is a chronic devastating muscular dystrophy with an incidence rate of 1 in 5000 boys and an average life expectancy below 30 years. Hypertrophic cardiomyopathy followed by end-stage dilated cardiomyopathy and heart failure are major factors in mortality in patients. Vamorolone is a recently discovered steroidal drug that represents a most promising treatment by simultaneously suppressing inflammation and cardiomyopathy in DMD patients while avoiding the safety concerns of the corticosteroid class. It acts on both glucocorticoid receptor (GR) and mineralocorticoid receptor (MR). We set to bridge the gap in knowledge of molecular mechanism of vamorolone acting on MR. Our cellular gene reporter assay showed that conventional drug prednisolone acts as an agonist for both MR and GR while vamorolone is a GR agonist but an MR antagonist. We have determined the X-ray crystal structure of the MR ligand binding domain (LBD)-vamorolone complex, revealing the lack of residue Ans770-mediated hydrogen bond compared to prednisolone binding. We identified a unique coregulator recruited by vamorolone when binding to MR, which potentially contributes to vamorolone-mediated MR antagonism. We have used differentiated cardiomyocytes from human DMD induced pluripotent stem cells and analyzed the transcriptional landscape changes using RNA-seq and ATAC-seq. These integrated studies lay the foundation for understanding how subtle chemical changes in ligands drive differential effects on a genome-wide scale and provide molecular insights for future MR antagonist design for better cardioprotection for DMD patients.