B29N(epsilon)-lithocholyl-gamma-L-beta Glu-desB30 human insulin [NN344] belongs to a group of insulins with fatty acid or sterol modifications. These insulin analogues have been found to form subcutaneous depots upon injection and hereby have a protracted release profile in vivo. In the present study, B29N(epsilon)-lithocholyl-gamma-L-Glu-desB30 human insulin was investigated using in-solution small-angle X-ray scattering (SAXS) at chemical conditions designed to mimic three stages during treatment in vivo: in-vial/pen, postinjection, and longer times after injection. We found that the specific insulin analogue formed a mixture of mono- and dihexamers under in-vial/pen conditions of low salt and stabilizing phenol. At postinjection, conditions mimicking a subcutaneous depot, B29N(epsilon)-lithocholyl-gamma-L-Glu-desB30 human insulin, formed very long straight soluble hexamer-based rods stacked along the Zn(II)-axis. The self-assembly was triggered by an increase in salt concentration when going from vial to physiological conditions. Mimicking longer times after injection and the additional removal of phenol caused the length of the rods to decrease significantly. Finally, we found that the self-assembly could be controlled by varying the amount of modification at the interaction interface by making mixed hexamers of B29N(epsilon)-lithocholyl-gamma-L-Glu-desB30 and desB30 human insulin. This opens extra possibilities for controlling the release profile of very-long-acting insulins.