Colloidal nanoparticles were prepared by aqueous self-assembly of amphiphilic β cyclodextrins (βCDs) acylated on their secondary face with C14 chains to a total degree of substitution of 7.0, via a thermolysin-catalyzed transesterification process. The small angle X ray scattering pattern of the nanoparticles was consistent with a reverse hexagonal organization. Cryo transmission electron microscopy images revealed particles with spectacular tortuous shapes and consisting of misoriented domains with a regular columnar hexagonal structure, separated by sharp interfaces. Edge dislocations as well as a variety of stepped tilt grain boundaries (GBs) composed of symmetrical and asymmetrical sections, together with one twist GB, were identified from axial views of the columnar organization. The tilt GB structure was analyzed using the concepts of coincidence site lattice and structural units developed to describe the atomic structure of interfaces in various types of polycrystals. The tilt GBs were described using sequences of βCD–C14 columns that differed by the number of neighboring columns (5, 6 or 7) and exhibiting distinctive contrasts. To our knowledge, this is the first time that these types of topological defects are described at the nanometric scale by direct observation of colloidal polycrystalline hexosomes of self-organized amphiphiles.