The effect of the smear layer on the penetration depth of three different root canal sealers into the dentinal tubules was examined in 64 recently extracted single-rooted teeth. After chemomechanical preparation, the samples were randomly divided in two equal groups. The smear layer remained intact in group A, whereas complete removal of the smear layer was performed in group B. Two roots from each group were used as controls. Ten roots from each group were obturated with laterally condensed gutta-percha points and sealers AH Plus, Apexit, and Roth 811, respectively. Examination in scanning electron microscope revealed that the smear layer obstructed all the sealers from penetrating dentinal tubules. In contrast, smear layer removal allowed the penetration of all sealers to occur to a varying depth. These findings suggest that smear layer plays an important role in sealer penetration into the dentinal tubules, as well as in the potential clinical implications.