The attachment of spindle fibres to the kinetochore is an important process that ensures successful completion of the cell division. The Ca2+ concentration increases during the mitotic phase and contributes microtubule stability. However, its role in the spindle organisation in mitotic cells remains controversial. Here, we investigated the role of Ca2+ on kinetochore fibres in living cells. We found that depletion of Ca2+ during mitosis reduced kinetochore fibre stability. Reduction of kinetochore fibre stability was not due to direct inhibition of microtubule polymerisation by Ca2+-depletion but due to elimination of one dynamic component of kinetochore, CENP-F from the kinetochore. This compromised the attachment of kinetochore fibres to the kinetochore which possibly causes mitotic defects induced by the depletion of Ca2+.