Skeletal muscle development and growth are regulated through multiple signaling pathways that include insulin-like growth factor I (IGF-I) and calcineurin activation of NFAT transcription factors. The developmental regulation and molecular mechanisms that control IGF-I gene expression in murine embryos and in differentiating C2C12 skeletal myocytes were examined. IGF-I is expressed in developing skeletal muscle and its embryonic expression significantly reduced in embryos lacking both NFATc3 and NFATc4. During development, the IGF-I exon 1 promoter is active in multiple organ systems, including skeletal muscle, whereas the alternative exon 2 promoter is expressed predominantly in liver. IGF-I exon 1 promoter flanking sequence includes two highly conserved regions that contain NFAT consensus binding sequences. One of these conserved regions contains a calcineurin/NFAT responsive regulatory region that is preferentially activated by NFATc3 in C2C12 skeletal muscle cells and NIH3T3 fibroblasts. This NFAT-responsive region contains three clustered NFAT consensus binding sequences, and mutagenesis studies demonstrate the requirement for two of these in calcineurin or NFATc3 responsiveness. Chromatin immunoprecipitation (ChIP) analyses demonstrate that endogenous IGF-I genomic sequences containing these conserved NFAT binding sequences interact preferentially with NFATc3 in C2C12 cells. Together, these studies demonstrate that an NFAT-rich regulatory element in the IGF-I exon 1 promoter flanking region is responsive to calcineurin signaling and NFAT activation in skeletal muscle cells. The identification of a calcineurin/NFAT responsive element in the IGF-I gene represents a potential mechanism of intersection of these signaling pathways in the control of muscle development and homeostasis.