The production of functional (nano)materials based on lignin as a renewable starting material depends on the thorough understanding of lignin’s physico-chemical properties, among which self-assembly and agglomeration/aggregation are the most important. Nevertheless, the knowledge about the structure-property relations for lignin is still in its infancy and needs further in-depth investigations. In this context, this works focuses on the study of the size and the colloidal stability of lignin nanoparticles (LNPs) prepared from softwood kraft lignin (SKL) using the solvent-antisolvent method. Conformational rearrangements of lignin chains were found to contribute significantly to the formation of the first lignin nuclei. The slow addition of ethylene glycol and THF into water caused the formation of nuclei with low aggregation numbers, minimizing the hydrodynamic volume of the final LNPs. On the other hand, a quick addition of these organic solvents created spatially and temporally higher lignin concentrations, yielding nuclei with high aggregation numbers and larger hydrodynamic volumes. Molecular dynamics simulations revealed the major role of intramolecular and intermolecular hydrogen bonds in this process, together with the contribution from π-π stacking interactions. The superficial concentration of phenolic and condensed guaiacyl units was found to strongly influence the corresponding LNPs’ zeta-potential values. Altogether, these results shed further light on the properties of colloidal lignin with a view to enabling its full potential as a key material for technological applications.