The copines are a family of C2‐ and von Willebrand factor A‐domain‐containing proteins that have been proposed to respond to increases in intracellular calcium by translocating to the plasma membrane. The copines have been reported to interact with a range of cell signalling and cytoskeletal proteins, which may therefore be targeted to the membrane following increases in cellular calcium. However, neither the function of the copines, nor their actual movement to the plasma membrane, has been fully established in mammalian cells. Here, we show that copines‐1, ‐2, ‐3, ‐6 and ‐7 respond differently to a methacholine‐evoked intracellular increase in calcium in human embryonic kidney cell line‐293 cells, and that their membrane association requires different levels of intracellular calcium. We demonstrate that two of these copines associate with different intracellular vesicles following calcium entry into cells, and identify a novel conserved amino acid sequence that is required for their membrane translocation in living cells. Our data show that the von Willebrand factor A‐domain of the copines modulates their calcium sensitivity and intracellular targeting. Together, these findings suggest a different set of roles for the members of this protein family in mediating calcium‐dependent processes in mammalian cells.Structured digital abstract  MINT‐8049236: Copine‐6 (uniprotkb:Q9Z140) and transferrin (uniprotkb:P02787) colocalize (MI:0403) by fluorescence microscopy (MI:0416)  MINT‐8049176: CD2 (uniprotkb:P06729) and Copine‐2 (uniprotkb:P59108) colocalize (MI:0403) by fluorescence microscopy (MI:0416)