This article shows that intramolecular hydrogen bonds formed at the three hydroxyl group positions of cellulosic glucose unit contribute in determining the handedness of chiral nematic mesophases ethylcellulose in CH2Cl2. Using two series of samples which differed only by increasing the ethyl substitution of the hydroxyl groups at either the C-6 or C-3 positions, the handedness of individual mesophase solutions was investigated using circular dichroism (CD). The CD spectra indicated that free hydroxyls at the C-6 position hindered dissolution in the solvent while hydroxyls at the C-3 position may play an important role in determining the handedness of the EC mesophase in CH2Cl2. Free hydroxyls at the C-3 position are known to easily form intramolecular hydrogen bonds, resulting in enhanced stiffness of the molecular chains. Therefore, these results lead to the conclusion that breaking intramolecular hydrogen bonds by increasing the substitution along the molecular chain causes it to become more flexible, which may account for the change in structural handedness.