High pressure and rotor/stator homogenisation are widely used techniques in modifying rheologically interesting polysaccharides such as cereal β-glucan for food purposes. However, the influence of the homogenisation-induced mechanical stress on β-glucan has not been reported. The influence of three different homogenisers (two high-pressure homogenisers and a rotor/stator homogeniser) was examined in terms of the change in flow and molecular properties of oat β-glucan in semi-dilute and concentrated solutions. A clear and irreversible decrease in viscosity and change in flow behaviour were observed after each homogenisation treatment of the semi-dilute solution. The viscosity had a linear relationship with molar mass in the high-pressure homogenised samples and they both decreased in parallel with the mechanical energy input. With the molar mass decrease, the shape of β-glucan became more spherical and dense. In addition, the molecular weight distribution narrowed and storage-related viscosity stabilized. No considerable differences between the influences of the two high-pressure techniques were observed inconsistently with previous studies. Both molecular and technical characteristics were concluded to affect the fragmentation of β-glucan in high-pressure homogenisation. Since homogenisation enhanced the structural stability of the solution and the fragmentation was dependent on the energy input, the techniques were concluded to be relevant methods for controlled fragmentation of β-glucan. Confocal scanning laser micrographs of oat β-glucan assemblies in 2 weeks stored aqueous solutions. Display omitted