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Background: The use of effective, low-cost, and easy-to-use products for early caries management will avoid loss of dental vitality and impairment in oral function. The ability of fluoride to re-mineralize dental surfaces has been widely reported as well as vitamin D demonstrated to have significant potential in improving the remineralization of early lesions on enamel surfaces. The aim of the present ex vivo study was to evaluate the effect of a fluoride and vitamin D solution in terms of formation of mineral crystals on the enamel of primary teeth, and their permanence over time on dental surfaces. Methods: Sixteen extracted deciduous teeth were cut to obtain 64 specimens that were divided into two groups. The first consisted of immersion of specimens for 4 days in a fluoride solution (T1); in the second group, the specimens were immersed for 4 days (T1) in fluoride and Vitamin D solution, and for a further 2 (T2) and 4 days (T3) in saline solution. Then, samples were morphologically analyzed by using Variable Pressure Scanning Electron Microscope (VPSEM) and underwent 3D surface reconstruction. Results: After a 4-day immersion in both solutions, octahedral-shaped crystals were formed on the enamel surface of primary teeth, demonstrating any statistically significant differences in terms of number, size, and shape. Moreover, the binding of the same crystals seemed to be strong enough to be maintained until 4 days in saline solution. However, a partial dissolution was observed in a time-dependent manner. Conclusions: A topical application of fluoride and Vitamin D promoted the formation of persistent mineral crystals on enamel surfaces of deciduous teeth and should be further studied to be potentially used as an alternative strategy in preventive dentistry.