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Objectives The present in vitro pH cycling study investigated potential differences between caries lesions created in fluorosed and sound enamel with regards to their responsiveness to fluoride under remineralizing conditions. Methods 360 human first molars (sound and fluorosed) were divided into four groups based on their Thylstrup–Fejerskov score (TF0-3). Each group was further divided into two treatment groups (n = 45): deionized water or 383 ppm fluoride. Artificial enamel caries lesions were created and pH cycled for 20 d using an established net remineralization model. Quantitative light-induced fluorescence was used throughout the study to investigate lesion severity and changes thereof. Data were analyzed using two-way ANOVA. Results There were no differences in lesion severity between all groups after lesion creation (plesion = 0.1934). The TF score vs. treatment interaction was significant at all other time points (p10 d = 0.0280; p20 d ≤ 0.0001; psecdemin = 0.0411). Relative differences in responsiveness to fluoride vs. deionized water increased with increasing TF scores. In comparison to lesions created in sound enamel, lesions created in enamel with moderate fluorosis (TF 2/3) were more prone to remineralization in the presence than in the absence of fluoride. Furthermore, lesions created in enamel with moderate fluorosis exhibited more remineralization in the presence of fluoride than lesions created in sound teeth, whereas the opposite was true for deionized water. Conclusion Bearing in mind the limitations of laboratory research, the extent of enamel fluorosis severity may directly impact subsequent lesion re- and progression as well as the lesion's responsiveness to fluoride. Clinical relevance Caries lesions in fluorotic teeth are more vulnerable to progression but respond more strongly to fluoride than those in non-impacted teeth.