Creatine kinase (CK) is a key enzyme to maintain the energy homeostasis in vertebrate excitable tissues. Due to its importance in cellular energetics, the activity and level of CK are crucial to cellular and body functions. CK is sensitive to oxidative stresses and is thought to be one of the main targets of oxidative modification in neurodegenerative diseases. In this research, we investigated the effect of copper, an essential trace element for all organisms and an inducer of the reactive oxygen species, on CK refolding. It was found that trace amounts of Cu(2+) (3mol eq of Cu(2+)) could efficiently block the refolding of CK. The Cu(2+)-trapped CK could not be reactivated by the addition of EDTA, but could be reactivated by DTT. Spectroscopic experiments suggested that copper ions blocked CK refolding by specifically binding with the monomeric refolding intermediate, which further retarded CK refolding and promoted the formation of off-pathway aggregates. The results herein suggested that Cu(2+)-induced CK dysfunction might be caused not only by the post-translational oxidation, but also by the direct binding of copper ions with the newly-synthesized polypeptides.