The gradient diffusion coefficients of recombinant human lactoferrin, a glycoprotein that is of commercial interest, have been extensively investigated theoretically and experimentally. A theoretical prediction was developed to allow calculation of the thermodynamic coefficient in terms of the electrostatic repulsive forces, London–van der Waals forces, entropic forces and additional interactions of unknown source and determination of the hydrodynamic coefficient by a perturbation theory method. In addition, dilute limit calculations were carried out for both of them. The thermodynamic and hydrodynamic coefficients were incorporated in the generalised Stokes–Einstein equation to calculate the gradient diffusion coefficient. Dynamic light scattering experiments were also conducted under different physiochemical conditions in which the gradient diffusion coefficients and the mean sizes of the macromolecules were measured. It was shown that of the theoretical approaches studied, good quantitative agreement between theory and experiment was found.