Four lipotetrapeptides derived from three glycines and one L-alanine were synthesized. They can self-assemble into twisted nanoribbons in a methanol/water mixture at a volume ratio of 6/4. With the movement of the L-alanine residue (namely, the shift of the methyl group) from the C- to the N-terminal, handedness inversion of the nanoribbons was observed. A molecular packing handedness inversion phenomenon was identified in the circular dichroism spectra. FT-IR and X-ray diffraction patterns indicated that the nanoribbons were constructed using lipotetrapeptide dimers. The electric dipole moment of the lipotetrapeptide dimer was proposed to dominate the handedness inversion of nanoribbons.