Pyrrole–imidazole polyamides (PIPs) bind to predetermined double-stranded DNA sequences and selectively target a large variety of DNA sequences. Although the forward-binding (5′-3′/N–C) orientation, in which the N-terminus of PIPs faces the 5′-terminus of DNAs, is considered to be the main binding manner of PIPs, a reverse-binding (5′-3′/C–N) orientation, in which the C-terminus of PIPs faces the 3′-terminus of DNAs, sometimes causes unintended binding. Here, we synthesized optical or structural isomers of previously reported cyclic PIPs (cPIPs), which differ in the position of the amino groups in the γ-turn units, and we investigated their binding affinities both in the forward- and reverse-binding orientation. We show that cPIPs with (R)-α-amino-γ-turn units prefer the forward orientation as do hairpin PIPs. More importantly, we document for the first time the remarkable reverse-binding preference of cPIPs with (S)-α-amino-γ-turns. These results indicate that the orientation preference of cPIPs can be controlled by the position of the amino groups on the γ-turn units, which may markedly increase the number of DNA sequences that can be targeted by PIPs.