We evaluated the efficacy of xylitol varnishes on the remineralization of newly erupted permanent and deciduous teeth in vitro and in situ. Human enamel specimens were randomly allocated to 8 groups (n = 15/group). Artificial caries lesions were produced and enamel alterations were quantified by surface/subsurface hardness and transverse microradiography. The blocks were then treated with the following varnishes: Duraphat TM ; 20 wt% xylitol (146 μm) varnish; 20 wt% milled xylitol (80 μm) varnish, and placebo varnish, and removed after 6 h of immersion in artificial saliva. In vitro: the blocks were subjected to pH-cycles for 8 days. In situ: fifteen subjects wore palatal appliances containing four pre-demineralized and treated enamel specimens, for 5 days. Data were statistically analyzed by ANOVA/ Tukey and Kruskal-Wallis/Tukey's test (p < 0.05). The %SHR in both studies was significantly increased by xylitol and Duraphat™ varnishes when compared to placebo. Considering subsurface remineralization, only the xylitol varnishes were able to significantly reduce the enamel lesion. Xylitol varnishes can be promising alternatives to promote enamel remineralization of newly erupted permanent and deciduous teeth.