Mammalian Twist1, a master regulator in development and a key factor in tumorigenesis, is known to repress transcription by several mechanisms and is therefore considered to mediate its function mainly through inhibition. A role of Twist1 as transactivator has also been reported but, so far, without providing a mechanism for such an activity. Here we show that heterodimeric complexes of Twist1 and E12 mediate E-box-dependent transcriptional activation. We identify a novel Twist1 transactivation domain that coactivates together with the less potent E12 transactivation domain. We found three specific residues in the highly conserved WR domain to be essential for the transactivating function of murine Twist1 and suggest an alpha-helical structure of the transactivation domain.