Objective: To evaluate the caries-protective effect of three recent dentin bonding agents (DBAs) on demineralization of root surface in vitro. Materials and Methods: The root surface of 70 freshly extracted caries-free human teeth was thoroughly cleaned and polished, thereby removing the cementum. The root surfaces were coated with acid-resistant nail polish and 2 mm × 3 mm rectangular window was prepared on the buccal surface. The samples were then divided randomly into three experimental groups; Group 1 (Xeno-V), Group 2 (Tetric N Bond), and Group 3 (Gluma Self-etch) which were further divided into two Subgroups (A and B) with 10 samples each in which second layer of adhesive was cured without air thinning and with air thinning respectively. Subsequently, all specimens were demineralized for 6 days with acidified gel (hydroxyethylcellulose, pH 4.8, 37°C). Each tooth samples were sliced in plano parallel section (80 ± 20 μm) by safe sided diamond disc and examined for caries-like lesions (demineralized area) under a polarized microscope. Data were analyzed using one-way ANOVA, Student's t- and Tukey honestly significant difference tests. Results: All control group specimen exhibited lesions with a mean depth of 86.15 μm. In Group 1–3, the lesion depth was reduced significantly, Subgroup A results were better than B. Conclusion: Demineralization on root surface can be impeded by DBA tested. Gluma Self-etch showed maximum caries-protective effect.