Euplotes octocarinatus centrin (EoCen) is a member of the EF-hand superfamily of calcium-binding proteins, which refer to nucleotide excision repair (NER). However, the role of centrin in NER is not clear. To explore the possible role of centrin, we initiated a physicochemical study of the N-terminal domain of Euplotes octocarinatus centrin (N-EoCen) with DNA in the absence or presence of Ca(II) in 10 mM Hepes at pH 7.4. N-EoCen shows unusual affinity for double-stranded DNA. The interaction results in the protein exposing more hydrophobic surface along with a certain perturbation taking place in the double helix structure. Interestingly, N-EoCen exhibits endonuclease-like activities via a hydrolysis pathway, which induces DNA strand breaks, such as supercoiled DNA into nicked circular and linear DNA. Importantly, mutation of serine (Ser) and threonine (Thr) to alanine (Ala) demonstrates that Ser and Thr, in particular Ser located at 22 (Ser22), may be the key residues responsible for DNA cleavage activity. The coordination of apoN-EoCen with Ca(II) can promote the binding to DNA and raise the cleavage activities. In contrast, the binding to Ca(II) of mutant proteins may trigger a conformational change so that the cleavage activity decreases dramatically, as confirmed by protein hydrolysis activity experiments. This is first report of the endonuclease-like activity of centrin, which provides valuable information for understanding a novel property of centrin, as well as knowledge of the functional diversity of centrin.