The cis-2-aminocyclohexanecarboxylic acid (cis-ACHC) oligopeptides with alternating chirality have been known to display both right- and left-handed 12/10-helical conformations in solution. We synthesized several cis-ACHC pentapeptides of which the third residue is substituted with a simple α-amino acid such as alanine, valine, and 2-aminoisobutyric acid (Aib). Then, we analyzed how introduction of the α-amino acid into the cis-ACHC pentapeptide can alter its folding structure by using the CD, 2D-NMR and the single-crystal XRD methods. Herein we demonstrate that introduction of D-alanine or D-valine can promote an intramolecular hydrogen bond to form a left-handed helical structure in this cis-ACHC pentapeptide. In contrast, introduction of L-alanine or Aib can interfere helical folding.