DNA integrity is challenged by both exogenous and endogenous alkylating agents. DNA repair proteins such as Escherichia coli AIkB family of enzymes can repair 1-methyladenine and 3-methylcytosine adducts by oxidative demethylation. Human AIkB homologue 5 (ALKBH5) is RNA N6-methyladenine demethylase and not known to be involved in DNA repair. Herein we show that ALKBH5 also has weak DNA repair activity and it can demethylate DNA 3-methylcytosine. The mutation of the amino acid residues involved in demethylation also abolishes the DNA repair activity of ALKBH5. Overexpression of ALKBH5 decreases the 3-methylcytosine level in genomic DNA and reduces the cytotoxic effects of the DNA damaging alkylating agent methyl methanesulfonate. Thus, demethylation by ALKBH5 might play a supporting role in maintaining genome integrity. (C) 2020 Elsevier Inc. All rights reserved.