The myocyte enhancer factor 2 (MEF2) family of transcription factors plays important roles in developmental processes and adaptive responses. Although MEF2 proteins are known to bind DNA in the nucleus to regulate specific gene expression, there are reports that show that MEF2 also functions in the cytoplasm. Previous structural studies of MEF2 focused exclusively on DNA-bound MEF2 with and without various corepressors or coactivators. While these studies have established a comprehensive structural model of DNA recognition and cofactor recruitment by MEF2, the structure of MEF2 not bound to DNA, which include cytoplasmic MEF2 and free MEF2 in the nucleus, is unknown. Here we determined the structure of the MADS-box/MEF2 domain of MEF2B without DNA nor cofactor. The Apo structure of MEF2B reveals a largely preformed DNA binding interface that may be important for recognizing the shape of DNA from the minor groove side. In addition, our structure also reveals that the C-terminal helix of the MEF2-specific domain could flip up to bind to the hydrophobic groove that serves as the binding sites of MEF2 transcription cofactors. These observations shed new insights into DNA binding and cofactor interaction by MEF2 proteins.