The novel transcription factor hMusTRD1alpha1 (human Muscle TFII-I-like Repeat Domain 1alpha1; previously named MusTRD1; (1)) was identified in a yeast one-hybrid screen as a protein that binds within an upstream enhancer-containing region (USE) of the skeletal muscle-specific gene, TNNI1 (human troponin I slow; hTnI( slow). It has been proposed that hMusTRD1alpha1 may play an important role in fiber-specific muscle gene expression by virtue of its ability to bind to an Inr-like element (nts -977 to -960) within the hTnI( slow) USE that is necessary for slow fiber-specific expression. In this study we demonstrate that both myocyte enhancer-binding factor 2C (MEF2C), a known regulator of slow fiber-specific genes, and hMusTRD1alpha1 regulate hTnI( slow) through the Inr-like element. Co-transfection assays in C2C12 cells and Cos-7 cells, demonstrate that hMusTRD1{allpha}1 represses hTnI( slow) transcription and prevents MEF2C-mediated activation of hTnI( slow) transcription. Gel shift analysis shows that hMusTRD1alpha1 can abrogate MEF2C binding to its cognate site in the hTnI( slow) enhancer. GST pulldown assays demonstrate hMusTRD1alpha1 can interact with both MEF2C and the nuclear receptor co-repressor (NCoR). The data supports the role of hMusTRD1alpha1 as a repressor of slow fiber-specific transcription through mechanisms involving direct interactions with MEF2C and NCoR.