Purpose: This study was designed to evaluate the effect of different concentrations of chitosan as final rinse on removing smear layer using scanning electron microscope (SEM) and to quantify the calcium content using energy dispersive X-ray microanalyzer (EDX). Materials and Methods: Fifty extracted human single rooted premolars were selected and instrumented with Revo-S Ni-Ti rotary files. Samples were divided into 4 experimental groups according to the final rinse used and control group (10 samples each) where group I,II,II,IV were irrigated with 0.2%,1%,2%chitosan and EDTA respectively ,Group V (Control group): Va (5 samples) irrigated with sterile saline. Vb (5 samples) not prepared or received any treatment. Samples were grooved and spilt longitudinally and smear layer were evaluated using (SEM) at (coronal, middle and apical) levels and calcium content of intraradicular dentin was measured using (EDX) at middle level. Results: Regarding smear layer removal, at the coronal level, there was a statistically significant difference among the tested groups and the control group in the mean scores of smear layer, however, there was no statistical significant difference among the tested groups in the mean scores of smear layer .At the middle and apical thirds, the lowest mean score of smear layer was recorded in specimens treated with 2% chitosan with a statistical significant difference from specimens treated with 0.2% chitosan. However, there was no statistical significant difference in the mean smear layer scores among 1% chitosan, 2% chitosan and 17% EDTA. Concerning calcium ion concentration there was no statistical significant difference among the tested groups. Conclusions: Chitosan was capable of adequately removing smear layer from intraradicular dentin when used as final rinse. The concentration of chitosan is influential in smear layer removal from intraradicular dentin, being more effective when used in 1% and 2% concentrations. Different concentrations of chitosan did not affect the calcium content of intraradicular dentin.