Objectives: The aim of this study was to determine the caries-protective effect of 3 different desensitizing agents (Seal & Protect 2.0, D/Sense 2, and Gluma Desensitizer) on root dentin in vitro. Method and materials: The root surfaces of 60 freshly extracted, caries-free human molars were used. After removing the cementum, the teeth were coated with an acid-resistant nail varnish, exposing 2 rectangular windows of 2 X 3 mm each on the root surface. One window served as an untreated control, and the other window was treated with 1 of the desensitizing agents. The specimens were randomly distributed among the following experimental groups: group A, D/Sense 2; group B, Seal & Protect 2.0; and group C, Gluma Desensitizer. Subsequently, all specimens were demineralized for 14 days with acidified gel (HEC, pH 4.8, 37°C). Two dentinal slabs were cut from each window. The slabs were ground to a thickness of 80 µm and immersed in water. The demineralization depth was determined using a polarized light microscope. Results: The nontreated control specimens showed lesions with a mean depth of 84.9 µm (± 6.0). In the specimens treated with the desensitizing agents, lesion depth was generally significantly reduced. Statistical analysis revealed significantly lower values for the specimens in group B in comparison with the others. Conclusion: Within the limitations of an in vitro investigation, it can be concluded that the demineralization of the root surface can be hampered by applying the desensitizing agents tested. Keywords: caries prevention, demineralization, dentin, dentin sensitivity, desensitizing agents, root caries