Abstract Objectives Caries progress might be controlled when collagen matrix could be preserved after demineralization. The aim of this pH cycling study was to investigate the effect of hesperidin, a citrus flavonoid antioxidant, on dentine collagen and remineralization in dentine lesion, and compared with that of chlorhexidine. Methods The pH cycling was employed on bovine root dentine by demineralization for 14 h, incubation in testing solutions (hesperidin or chlorhexidine) for 2 h and remineralization with bacteria-derived collagenase for 8 h, for 8 days. Calcium release was measured by means of an atomic absorption spectrophotometer, and degraded collagen matrix by collagenase was investigated by assaying hydroxyproline. The lesion depth and mineral loss was evaluated by means of transverse microradiography. Results The effect of testing solutions had a significant difference on the results of chemical analyses ( p < 0.0115 for calcium release; p < 0.0008 for degradated collagen). The lesion depth and mineral loss were reduced in the lesions where were incubated with hesperidin and chlorhexidine. The remineralization in deep lesions was found when the matrix was incubated in hesperidin, whilst no mineral uptake in deep lesion when incubated in chlorhexidine. Conclusion Hesperidin preserved collagen and inhibited demineralization, and enhanced remineralization even under the fluoride-free condition.