Fe(II)/2OG-dependent dioxygenases of the AlkB family catalyze a direct removal of alkylated damages in the course of DNA and RNA repair. A human homolog of the E. coli AlkB ALKBH3 protein is able to hydroxylate N1-methyladenine, N3-methylcytosine, and N1-methylguanine in single-stranded DNA and RNA. Due to its contribution to an antitumor drug resistance, this enzyme is considered a promising therapeutic target. The elucidation of ALKBH3’s structural peculiarities is important to establish a detailed mechanism of damaged DNA recognition and processing, as well as to the development of specific inhibitors. This work presents new data on the wild type ALKBH3 protein and its four mutant forms (Y143F, Y143A, L177A, and H191A) obtained by circular dichroism (CD) spectroscopy. The dataset includes the CD spectra of proteins measured at different temperatures and a 3D visualization of the ALKBH3–DNA complex where the mutated amino acid residues are marked. These results show how substitution of the key amino acids influences a secondary structure content of the protein.