The aim was to investigate the anti-caries properties of calcium glycerophosphate (CaGP) using an in vitro bacterial flow cell model. Four flow cells, inoculated from a chemostat containing a seven-organism bacterial consortium, were pulsed with sucrose twice daily, to provide an acidic challenge and pH-cycling conditions. Blocks of enamel and dentine were mounted in each flow cell. In a study on the effect of CaGP concentration, CaGP was pulsed into three of the flow cells, at the same time as the sucrose, to give concentrations of 0.10, 0.25 and 0.50%. Water was pulsed into the fourth flow cell with the sucrose. Microradiography revealed a significant dose response of decreasing demineralization as CaGP concentration increased. Reductions at 0.25 and 0.5% were significant when compared to the control. A second study investigated the effect of timing of CaGP pulsing, relative to sucrose, on enamel and dentine demineralization. CaGP (flow cell concentration 0.2%), was pulsed 1 h before, during or 1 h after the sucrose pulse; a water control was employed. In enamel, pulsing CaGP before the sucrose reduced demineralization significantly compared to concurrent pulsing, which in turn gave a significant reduction compared to pulsing after sucrose, which did not reduce demineralization significantly compared to the water control. In dentine, CaGP reduced demineralization significantly only when pulsed before the sucrose. The findings suggest that in vivo, the anti-caries potential of CaGP may be greater if it is applied before a cariogenic challenge.