The ubiquitously expressed RAPGEF1(C3G), regulates differentiation, and is essential for development of mouse embryos. While multiple transcripts have been predicted, evidence of their expression and function is scarce. We demonstrate tissue and development specific expression of novel transcripts with exons 12-14 in various combinations, in the mouse. These exons encode an intrinsically disordered serine-rich polypeptide, that undergoes phosphorylation. Isoform switching occurred during differentiation of myoblasts and mouse embryonic stem cells. In silico structure and docking studies indicated that the additional exons alter intra-molecular interactions keeping it in a closed confirmation, and interaction with its target, RAP1A. Our results demonstrate the expression of novel RAPGEF1 isoforms, and suggest cassette exon inclusion as an additional means of regulating RAPGEF1 activity during differentiation.