Native collagen molecules usually contract upon dehydration, but the details of their interaction with water are poorly understood. Previous molecular modeling studies indicated a spatially inhomogeneous response, with a combination of local axial expansion and contraction. Such sequence-dependent effects are difficult to study with native collagen. In this article, we use collagen-mimetic peptides (CMPs) to investigate the effect of osmotic pressure on several collagen-mimetic sequences. Synchrotron x-ray diffraction combined with molecular dynamics simulations shows that CMPs pack differently depending on osmotic pressure and exhibit changes in the helical rise per residue of individual molecules. Infrared spectroscopy reveals that osmotic pressure affects the stability of the triple helix through changes in triple helix-stabilizing hydrogen bonds. Surprisingly, CMPs with the canonical collagen sequence glycine–proline–hydroxyproline are found to elongate upon dehydration, while sequence modifications are able to reverse this tendency. This strongly suggests that the overall contraction of native collagen molecules is not programmed into the canonical sequence but is specific to local amino acids that substitute for proline or hydroxyproline along the protein chain. Collagen is an essential protein in mammalian extracellular tissues and a better understanding of its mechanical function is important both from a materials science and from a biomedical viewpoint. Recently, collagen has been shown to contract along the fibre direction when subjected to osmotic stress, a process that could play important roles in strengthening bone and in developing tissue tension during extracellular matrix development. The present work uses collagen-like short peptides to show that the canonical collagen sequence is not responsible for this contraction. The conclusion is that the collagen amino acid sequence must have evolved to include guest sequences within the canonical glycine-proline-hydroxyproline repeat that provide the observed contractility.