Background: The aim of this study was to evaluate the effect of a desensitizing agent on microleakage of Class V cavities. Material and Methods: 72 premolar teeth were used. There were 6 groups. Class V restorations were prepared with two different restorative materials (Equia fil, GC, America and Grandio, VOCO, Germany) and two adhesive systems (Clearfil SE Bond, Kuraray, Japan and S3 Bond Plus, Kuraray, Japan) with and without desensitizing agent (Gluma Desensitizer, Heraeus Kulzer, Germany). Restorations were polished with aluminum oxide abrasive discs. Then a range of 5 - 55C thermocycling was performed 10.000 times. The microleakage of restorations was examined with dye penetration method (Basic fuchsine). Bonferroni corrections and Kruskal-Wallis test were used to determine the significance of differences in occlusal and gingival dye penetration scores between groups. Results: There was no stastistical significance between the occlusal and gingival microleakage scores within the groups were shown. Conclusions: It can be concluded that use of desensitizing agent under both high viscosity glass ionomer restorative materials and resin composites doesn’t affect the microleakage.