Results of an investigation of the interaction potential of synthetic and pre-treated calcium silicate hydrate (C-S-H) [with hexadecyltrimethylammonium (HDTMA)] are reported. The effective and strong interaction of these molecules with the C-S-H surface was shown using 13C and 29Si cross polarization magic angle spinning (CP MAS) nuclear magnetic resonance, x-ray diffraction, thermogravimetric analysis, scanning electron microscopy, and Fourier transform infrared spectroscopy analysis. The HDTMA–C-S-H interaction is influenced by the poorly crystallized layered structure of C-S-H. An indefinite number of layers and an irregular arrangement are confirmed by the SEM images. The position and shape of the 002 reflection of C-S-H are affected by drying procedures, chemical pre-treatment, and reaction temperature. Recovery of the initial 002 peak position after severe drying and rewetting with distilled water or interaction with HDTMA is incomplete but accompanied by an increase in intensity. It is inferred that the stability of C-S-H binders in concrete can be affected by a variation in nanostructure resulting from engineering variables such as curing temperature and use of chemical admixtures.