Aim: The aim of the present study was to study the effect of smear layer removal on the penetrability and sealing ability of a new root canal sealer. Methodology: One hundred and one human single rooted teeth were used for this study. Eighty specimens were used for the sealing assessment while, the other twenty one were used for the penetrability assessment. For sealing assessment twenty specimens were equally divided into negative and positive control groups and sixty specimens were also equally divided for six experimental groups where n=10. For penetrability assessment eighteen specimens were equally divided into six experimental groups where n=3 while the remaining three were used as control group. In the smear layer-removal groups, smear layer was removed with 3 mL of 5.25% NaOCl followed by 3 ml of 17% EDTA (pH 7.7) for 1 minute and finally a solution of 3 ml saline was used as a final rinse. In the smear layer-keeping groups, 3 mL of 5.25% NaOCl was used alone. Sealing ability was assessed using dye extraction method while, the penetrability was assessed using scanning electron microscope. Results: results showed that there was a statistically significant difference between the smear layer removal groups and smear layer keeping groups for the three sealers as for their sealing ability while, in the penetrability assessment results showed that there was a statistically significant difference regarding the resin based sealers but not for the bioceramic sealer. Conclusion: It was concluded that both Resin based sealers and Silicone based sealers showed good sealing ability while, Resin based sealers showed better penetration than silicone based sealers.