Axin interactor, dorsalization‐associated (Aida) was identified as a regulatory factor that utilizes its C‐terminal region to interact with axis formation inhibitor (Axin). Aida abrogates the Axin‐mediated Jun N‐terminal kinase activation required for proper dorsalization during zebrafish embryonic development, and thus functions as a proventralization factor. Here, we report the structure of Aida C‐terminal fragments, which adopt a conventional C2 domain topology. We also demonstrate that Aida can specifically bind to phosphoinositides in a Ca2+‐independent manner, and is able to associate with the cell membrane via a novel positively charged surface, namely a basic loop. Mutation of the positively charged patch on the basic loop leads to destabilization of the Aida–membrane association or disruption of the Aida–Axin interaction, resulting in impaired Jun N‐terminal kinase inhibition. Together, our findings provide a molecular basis for C2 domain‐mediated Aida–membrane and Aida–Axin associations.DatabaseThe atomic coordinates and structure factors of the mouse Aida C2 domain (code: 2QZ5) and the zebrafish Aida C2 domain (code: 2QZQ) have been deposited in the Protein Data Bank (http://www.rcsb.org/)Structured digital abstract  AIDA physically interacts with Axin by anti tag coimmunoprecipitation (View interaction)