Formation of magnetite nanocrystals by magnetotactic bacteria is controlled by specific proteins which regulate the particles' nucleation and growth. One such protein is Mms6. This small, amphiphilic protein can self-assemble and bind ferric ions to aid in magnetite formation. To understand the role of Mms6 during in vitro iron oxide precipitation we have performed in situ pH titrations. We find Mms6 has little effect during ferric salt precipitation, but exerts greatest influence during the incorporation of ferrous ions and conversion of this salt to mixed-valence iron minerals, suggesting Mms6 has a hitherto unrecorded ferrous iron interacting property which promotes the formation of magnetite in ferrous-rich solutions. We show ferrous binding to the DEEVE motif within the C-terminal region of Mms6 by NMR spectroscopy, and model these binding events using molecular simulations. We conclude that Mms6 functions as a magnetite nucleating protein under conditions where ferrous ions predominate. Biomimetic synthesis: Mms6 is a key protein involved in the formation of magnetite nanocrystals by magnetotactic bacteria. To understand the role of Mms6 during in vitro iron oxide precipitation, in situ pH titrations (see figure), NMR spectroscopy, and molecular simulations were performed. The results suggest that Mms6 functions as a magnetite nucleating protein under conditions where ferrous ions predominate.