VASP is an actin polymerization regulatory protein, and is a founding member of the Ena/VASP family of proteins. The EVH1 domain, a conserved component of the Ena/VASP family, localizes these proteins to sites of high cytoskeletal dynamics through its selective binding to the sequence motif (F/W)PxhP, where x is any residue and h is an aliphatic residue. Our NMR studies of VASP-EVH1 interactions with the innate immunity signaling protein IRAK1 and with the cytoskeletal protein zyxin have revealed an unexpected mechanism of regulation and an unexpected mode of binding, respectively. These results provide important insights into the transient subcellular localization of VASP in response to innate immunity signaling, and to a novel binding mode that could provide an advantage for specific partners that contain sequential binding motifs.