Protein–DNA recognition plays a crucial role in gene expression and regulation. In this work, we have analyzed the influence of cation–π interactions to the stability of 62 protein–DNA complexes. A new criterion has been formulated to delineate the cation–π interactions based on (i) the distribution of atoms in the π system (5 and 6-member rings) of DNA bases around the positive charged atoms of Lys and Arg and (ii) the energetic contribution of contacting atoms from electrostatic and van der Waals interactions. Our method shows the presence of cation–π interactions in 92% of the complexes. The side chain of Arg is more likely than that of Lys to be in cation–π interactions. In both Lys and Arg, the cationic groups have stronger cation–π interaction energy than the atoms with effective positive charge. The aromatic chains of purines (A and G) are exhibiting more cation–π interactions than pyrimidines (C and T). The Arg-G pair has the strongest interaction energy of −4.3 kcal/mol among all the possible pairs of amino acids and bases. The interaction energy is always positive for T and we observed few favorable interactions with C. Further, we found that the cation–π interactions due to 5-member rings of A and G are stronger than that with the atoms in 6-member rings. The distribution of base atoms around the charged atoms shows that the N7 in the 5-member ring of G is making significant number of close contacts with NZ of Arg, which is important to establish dominant cation–π interactions.