The alpha-SG promoter is composed of a plethora of cis-regulatory elements, whose individual contribution to alpha-SG gene expression modulation remains unknown. We have identified a negative regulatory element in the alpha-SG distal promoter including two conserved E-boxes (E1 and E2), which interact with MyoD. We found that E1 and E2 negatively modulate the transactivation potential of MyoD on the alpha-SG core promoter. Moreover, such negative effect is mainly mediated by E2, which is surrounded by conserved nucleotides conferring MyoD binding capacity. Our results suggest that modulation of MyoD activity by E1, and particularly E2, contributes to the negative regulation of alpha-SG gene expression during myogenic differentiation.