Polyphenol released from food matrices is the first stage for their potential beneficial effects on human health. To better understand how natural barriers such as plant cell membranes and cell walls modulate polyphenol release, the major phenolic compounds within cells in apple pieces were directly localized, and their release under different thermal processing and acidic digestion conditions measured. The plasma membrane was found to be more thermally stable than the tonoplast, with membrane disruption occurring above 60 °C after processing for more than 10 min, acting as an efficient trigger for increased polyphenol release from 15% to more than 50%. Confocal microscopy of phenolic compounds in apple cells after thermal processing showed a clear relocation from uniform distribution in vacuoles to localization around cell walls, suggesting that the non-released polyphenols were cell wall associated. No additional polyphenols were released as a result of acidic conditions (pH 2-5) likely to be encountered in the stomach. Processing (thermal, pH) promoted polyphenol release by disrupting intracellular barriers, thus increasing the contact with cell walls and modulating bioaccessibility by controlling the interactions between cell walls and polyphenols.