The impact of high intensity ultrasound (US, 45 and 20 kHz) on a purified macromolecular fraction (more than 85% of polymeric procyanidins) from grape seed extract was investigated. Matrix-Assisted Laser Desorption/Ionisation (MALDI-TOF), Reverse Phase High Performance Liquid Chromatography (RP-HPLC) and Fourier-transform infrared spectroscopy (FTIR) revealed a modification in the chemical structure of these macromolecules treated by US and, particularly, bath US produced a considerable increase of up to 49, 41 and 35%, respectively, of catechins and oligomeric and polymeric procyanidin contents of the treated purified fraction. Bath US also produced an important increase in the number of procyanidins with higher molecular mass (up to decamers) and an overall increase in the mass signal intensities in most of the detected B-type procyanidin series, as well as an important increase of the antioxidant activity of the macromolecular fraction of procyanidins. These results could be ascribed to a certain disaggregation of procyanidins linked to other biopolymers, such as proteins and/or polysaccharides, indicating that US is an efficient technology to modify the chemical structure and hence the bioactivity of tannins.