Otoferlin (Otof), whose genetic mutations cause profound deafness in humans, is a protein composed of at least six C 2 domains, which are known as Ca 2 + -binding and phospholipid-binding regions. Mammalian ferlin proteins are proposed to act in membrane fusion events, with Otof being specifically required for exocytosis in auditory hair cells. Ferlin C 2 domains exhibit a rather low level of sequence similarity to those of synaptotagmins, protein kinase C isoforms, or phospholipases. Here, we report the crystal structure of the N-terminal C 2 domain of Otof (C 2 A) at 1.95-Å resolution. In contrast to previous predictions, we found that this C 2 domain is complete with eight β-strands. Comparing the structure of Otof C 2 A to those of other C 2 domains revealed one top loop in Otof to be significantly shorter. This results in a depression of the surface, which is positively charged for the Otof C 2 A domain, and contrasts with the head-like protrusion surrounded by a negatively charged “neck” typically found in other C 2 domains. Isothermal titration calorimetry and circular dichroism spectroscopy studies confirmed that Otof C 2 A is unable to bind Ca 2+ , while the synaptotagmin-1 C 2 A domain exhibited Ca 2+ binding under the same conditions. Furthermore, floatation assays revealed a failure of Otof C 2 A to bind to phospholipid membranes. Accordingly, no positively charged β-groove-like surface structure, which is known to bind phosphatidylinositol-4,5-bisphosphate in other C 2 domains, was found at the respective position in Otof C 2 A. Taken together, these data demonstrate that the Otof C 2 A domain differs structurally and functionally from other C 2 domains.