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Introduction: Our study evaluated the impact of sodium thiosulfate (ST) irrigation, subsequent to sodium hypochlorite (NaOCl) and just before root canal filling, on the filling quality (interfacial adaptation and penetration segment) of an epoxy resin-based root canal sealer. Methods and Materials: Twenty single-rooted human teeth were prepared with the ProTaper system. The specimens were then divided into the following groups: 5.25% NaOCl irrigation (NaOCl group) and 5.25% NaOCl irrigation+0.5% sodium thiosulfate (NaOCl+ST group). The root canals were filled using single-cone technique with ProTaper F3 cones and AH-Plus sealer, labeled with rhodamine B dye to allow analysis under a confocal laser scanning microscopy (CLSM). All samples were sectioned at 2, 4, and 6 mm from the apex and prepared for CLSM analysis. The percentage of voids, gaps and dentinal sealer penetration segment of the canal were calculated at the apical, middle and coronal thirds. The non-parametric Mann-Whitney statistical test was used at 5% significance level. Results: Higher percentage of gaps and voids were observed at all root thirds of the NaOCl group when compared to the NaOCl+ST group (P<0.05). There was a significant increase in the penetration segment of NaOCl+ST group at the coronal and middle root third when compared to the NaOCl group (P<0.05). Conclusion: Our in vitro results showed that the use of ST as an antioxidant agent after NaOCl irrigation promoted a better interfacial adaptation and penetration [ABSTRACT FROM AUTHOR]