Cyclic α,α-disubstituted amino acids (dAAs) are powerful tools for stabilizing peptide helices, yet the effects of ring size on helical conformation remain poorly understood. Here, we report the design, synthesis, and structural analysis of template peptides incorporating three cyclic dAAs─1-aminocyclopropane-1-carboxylic acid (Ac3c), 1-aminocyclobutane-1-carboxylic acid (Ac4c), and 1-aminocyclopentane-1-carboxylic acid (Ac5c). Circular dichroism (CD) and infrared (IR) spectroscopy demonstrated that oligomers containing Ac3c-Ac5c residues adopt stable helical structures in solution. X-ray crystallographic studies further revealed the first solid-state structures of Ac3c- and Ac4c-containing peptides, showing well-defined right-handed helices stabilized by intramolecular i → i + 3 and i → i + 4 hydrogen bonds. Comparative analysis highlighted that ring strain in Ac3c weakens its helicogenic effect relative to those of Ac4c and Ac5c, underscoring the importance of ring size in dictating backbone torsion angles and hydrogen-bonding networks. To probe functional implications, template peptides were incorporated into cell-penetrating peptide conjugates (Block3 derivatives). While Ac4c- and Ac5c-containing derivatives retained helicity and supported siRNA internalization, the Ac3c analogue adopted a random conformation and lost activity. These findings establish cyclic dAAs as versatile helix-inducing modules, provide new structural insights into ring-size-dependent helix stabilization, and suggest design principles for peptide-based foldamers and delivery systems.