Chitosan with 91.7% deacetylation was hydrolysed by a commercially available and efficient neutral protease. The degradation was monitored by gel permeation chromatography. Factors affecting the enzymatic hydrolysis of chitosan were studied. The structures of degraded chitosans were characterised by X-ray diffraction, Fourier transform infrared and MALDI-TOF mass spectrometry. Classical Michaelis–Menten kinetics parameters were measured by analysis of reducing sugars. The neutral protease showed optimum depolymerisation at pH 5.4 and 50 °C. Different reaction times gave chitosan with different molecular weights. Mn2+ was the most efficient activator of the enzymatic reaction. The enzymatic hydrolysis was endo-action and mainly occurred in a random fashion. The degree of deacetylation of the main hydrolysis products decreased compared with the initial chitosan. The decrease of molecular weight led to transformation of crystal structure and the increase of water solubility, but the chemical structures of residues were not modified. The degree of polymerisation of chito-oligomers was mainly from 3 to 8.