Nucleobindin‐1 (NUCB1) and nucleobindin‐2 (NUCB2) are multifunctional proteins that interact with Ca2+, nucleic acids and various regulatory proteins in different signaling pathways. So far, our understanding of the regulation of the biological functions of nucleobindins remains limited. In our proteome‐wide selection for downstream caspase substrates, both NUCB1 and NUCB2 are found to be the downstream substrates of caspases. We report here the detailed analyses of the cleavage of nucleobindins by caspases. Significantly, the caspase cleavage sites are located exactly at one of the Ca2+‐binding EF‐hand motifs. Our results suggest that the functions of nucleobindins could be modulated by caspase‐mediated cleavage in apoptosis.