This study investigated the effects of a new strontium-doped bioactive glass and fluoride on hydroxyapatite crystallization. We designed an in vitro experiment with calcium phosphate (CaCl2·2H2O + K2HPO4 in buffer solution) with different concentrations of strontium-doped bioactive glass (1 mg/mL or 5 mg/mL), and different concentrations of fluoride (0 ppm, 1 ppm or 5 ppm). Tris-buffered saline served as negative control. After incubation at 37 ℃ for 48 h, the shape and organization of crystals were examined by transmission electron microscopy (TEM) and electron diffraction. Structure of the crystals was assessed by powder X-ray diffraction (P-XRD) and unit cell parameters were calculated. Characterization of the crystals were performed by Raman spectroscopy and Fourier Transform Infrared Spectroscopy (FTIR). TEM and selected-area electron diffraction revealed that the precipitates in all experimental groups were crystalline apatite. There was an interaction between strontium and fluoride with different concentrations on crystal thickness (p = 0.008). P-XRD indicated the formation of strontium-substituted-fluorohydroxyapatite and strontium-substituted-hydroxyapatite in the groups with both bioactive glass and fluoride. Expansion or contraction of crystal unit cell was influenced by the concentrations of strontium and fluoride. Raman spectra showed strong phosphate band at 960 cm-1 in all experimental groups and displayed no obvious shift. FTIR results confirmed the formation of apatite. The results of this study suggest that strontium-doped bioactive glass and fluoride have synergistic effects on hydroxyapatite crystallization. Strontium-doped bioactive glass and fluoride have synergistic effects on hydroxyapatite crystallization by producing strontium-substituted-hydroxyapatite and strontium-substituted-fluorohydroxyapatite with enhanced bioactivity and reduced solubility which could be beneficial for caries management. Copyright © 2021 Elsevier Ltd. All rights reserved.