Using intrinsic protein fluorescence spectroscopy actin-regulating protein villin domain 6 calcium-binding properties were characterized. Acquired data suggests the presence of calcium-binding site in villindomain 6. The affinity of this calcium-binding site was estimated to be 32 µM, which suggested that the domain could be responsible for physiological transition of villin from bundling to severing actin filaments in response to the increase of intercellular calcium concentration.