Background: Dentin hypersensitivity (DH) affects all age groups in a population and is perceived as pain to any stimuli. From time immemorial, researchers have sought herbal or natural solutions to treat hypersensitivity. Propolis is one such natural, nontoxic resinous substance produced by honey bees, which is useful in various applications in dentistry and effective in treating dentinal hypersensitivity. Aim: The aim of this in vitro study is to assess the effect of propolis varnish on occlusion of dentinal tubules thus aiding in the treatment of dentinal hypersensitivity. The objective is to evaluate the effectiveness of the proposed treatment using scanning electron microscope (SEM) imaging. Materials and Methods: Twenty naturally extracted teeth were collected and stored until in vitro assessment. Discs obtained from each tooth were divided into two groups of 20 each - I (test) and II (control), with each tooth acting as its own control. Propolis varnish was applied only to the acid-etched surface of the exposed dentin of discs in the test group, whereas all the discs were subjected to SEM imaging. Results: Statistical analysis showed a significant reduction in open tubules (P < 0.001) from 160 ± 6.97 before treatment to 61.20 ± 9.10 after propolis varnish application in the test group. Conclusion: This study showed the promise of propolis varnish as a natural treatment modality for DH. © 2019 Indian Journal of Dental Research | Published by Wolters Kluwer - Medknow.