Significance Actin is one of the most abundant proteins in eukaryotic cells. Actin filaments together with a large number of actin-binding proteins are critical players in many cellular functions, ranging from cell motility and muscle contraction to maintenance of cell shape and transcription regulation. α-Actinin—a member of the spectrin superfamily—is an archetypical F-actin–binding and –bundling protein. It is known that Ca 2+ inhibits α-actinin capacity to bundle F-actin. We present a structure of a Ca 2+ -regulated α-actinin and propose the mechanism for its regulation. We uncover that Ca 2+ binding triggers an increase in protein rigidity, leading to reduced conformational flexibility and bundling activity. The proposed molecular mechanism is likely to be a blueprint for regulation of spectrin-like proteins.