Dentine caries is a process of demineralization and subsequent degradation of the collagenous matrix. Host‐derived proteolytic enzymes, such as matrix metalloproteinases (MMPs), play a role in this process of dentine collagen degradation. Hampering this degradation retards the caries process. Dietary antioxidants, such as the flavonoid hesperidin, can inhibit the proteolytic activity of MMPs and act as natural stabilizers of collagen. The aim of this study was to investigate the anti‐collagenolytic activity of hesperidin in an in situ model. A single‐blind, split‐mouth, in situ experiment was designed. Seventeen participants received two completely demineralized dentine specimens placed contralaterally in the buccal flanges of their partial prosthesis. During the 4‐wk experimental period, the participants immersed the dentine specimens in a test solution [1,000 parts per million (p.p.m.) hesperidin] or a control solution (saline), twice daily for 3 min. After the in situ period, the specimens were retrieved and their collagen content was determined. A saliva sample was taken at the start and at the end of the experimental period, to assess collagenolytic activity. A significant protection of collagen, of 24%, was observed in the hesperidin‐treated specimens compared with the control‐treated specimens. No correlation was found between salivary collagenolytic activity and loss of collagen in the control‐treated specimens. The results of this in situ study show that hesperidin could play a role in the preservation of dentine collagen matrix.