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Objective: Our aim was to compare acquired acid resistance in dental enamel after Er:YAG and CO₂ laser irradiation in vitro with additional fluoride treatment. Background data: The application of lasers in dental hard tissue can impact dental constituents and fluoride with complex interactions. Methods: Bovine enamels were divided into four groups according to their surface condition: unlased, Er:YAG laser-ablated, fluoridetreated after Er:YAG laser ablation, and CO₂ laser-irradiated after Er:YAG laser and fluoride treatment. Calcium distribution in enamel after pH-cycling process and crystallographical change after laser treatment were evaluated. Results: The crystallinity of enamel was much improved after Er:YAG laser ablation. The CO₂ laser irradiation in the fluoride-treated laser enamel formed α-TCP and fluorapatite. The change of calcium distribution in enamel was least in the CO₂ laser-irradiated specimens; the fluoride-treated specimen after Er:YAG laser ablation was next. Additional fluoride treatment both after Er:YAG and before CO₂ laser irradiation improved the acid resistance of enamel. Conclusion: Both external treatment of fluoride and irradiation of CO₂ laser after Er:YAG laser ablation greatly reduced calcium loss in enamel during the pHcycling process.