Insulin-like growth factor 1 (IGF-I) expression is subject to complex temporal and spatial regulation. Endocrine synthesis occurs in the liver, where transcription is initiated from promoters located in either exon 1 (P1) or in exon 2 (P2), whereas local transcription is mainly initiated from P1. IGF-I is expressed in a range of tissues and, in particular, it is an important regulator of skeletal muscle mass, although mechanisms of tissue-specific regulation remain to be fully characterized. Gene regulation in skeletal muscle is associated with the E-box DNA element (CANNTG) recognized by myogenic regulatory factors (MRFs), such as MyoD1. Transcription element profiling identified a hypothetical myogenic E-box (sequence CAGCTG) within P1, immediately upstream of the major muscle transcriptional start site, and we sought to test its activity in differentiating C2C12 myoblasts. We found P1-driven IGF-I mRNA expression to be associated with myogenic differentiation and, moreover, that a single base-pair mutation in the E-box specifically reduced expression in myofibers. A synthetic enhancer construct containing a triplet repeat of the E-box was active in muscle cells and strongly induced in myofibers. The capacity of a double-stranded IGF1 E-box probe (but not one bearing a single base pair alteration) to bind C2C12 nuclear lysates increased with myogenesis and a transactivation assay demonstrated that the E-box was recognized by E-protein:MRF heterodimers. Mechanisms of tissue-specific gene activation are of increasing biological interest and we have identified a cis-element able to direct muscle-specific IGF1 gene expression.