Self-assembling peptides are promising biomaterials for spinal cord repair as they can easily be injected into the lesion site and can provide physical support to regrowing nervous tissue. However, to improve upon the design of synthetic scaffolds for spinal cord injury, characteristics of the scaffold/host relationship need to be further investigated. In the current study we aimed to evaluate both the mechanical properties and the therapeutic effect of two self-assembling peptides B24 and biotin-LDLK12 in spinal cord injury. Atomic force microscopy and rheology were used to characterise various concentrations of the two peptides in terms of the propensity to form nanostructures and the viscoelastic properties. Concurrently, these peptide solutions were injected into the contused spinal cord of rats to evaluate both diffusibility within the tissue, and scaffold formation in vivo. After selection of the best concentration for delivery in vivo, the two self-assembling peptides were tested in the contused spinal cord of rats for their influence on hematoma and cyst formation, biocompatibility and permissiveness for axonal growth. The results suggest that rheology can provide a useful indication to predict the hydrogel formation and diffusibility of the self-assembling peptides in vivo. Moreover at three days post-injury both self-assembling peptides had a good hemostatic effect and at 28 days they improved axon regrowth. In summary, the injectable self-assembling hydrogels could attenuate hematoma and provide a therapeutic effect in a spinal cord injury model.