Purpose: To evaluate the microtensile bond strength (µTBS) of resin sealer on enamel substrates after cariogenic challenge. Materials and Methods: Enamel blocks were obtained from human third molars and randomly divided into 6 groups (n = 10) according to enamel substrates (S: sound, CL: caries-like lesion, or CLTF: caries-like lesion + topical fluoride application) and sealant material (F: FluroShield, or H: Helioseal Clear Chroma). Sealants were placed on enamel surfaces, stored in 100% humidity (24 h, 37°C), and longitudinally sectioned into hourglass shapes. According to the groups, pH cycling was applied and the µTBS test was performed. The fracture patterns were assessed by SEM. Results: Regarding substrates, the highest µTBS values in MPa were observed for CLTF enamel (26.0 ± 7.6), followed by S (22.0 ± 7.4) and CL (15.5 ± 4.9). A significant interaction was found between material and pH cycling (p = 0.0395). F (23.9 ± 7.6) showed higher µTBS values than H (18.3 ± 7.5) when submitted to pH cycling. The majority of samples presented mixed failure. Conclusions: Enamel substrate significantly affected µTBS, with the highest values for remineralized caries-like enamel lesions. Furthermore, µTBS values were dependent on both materials and pH cycling. Keywords: preventive therapy, microtensile bond strength, fissure sealing, enamel, caries-like lesion, cariogenic challenge model