Background and Aim: The purpose of this study was to evaluate and compare the cytotoxicity of three dentin bonding systems, (4th, 5th and 6th generation of DBAs) on L929 cell line.Materials and Method: Thirty human premolar teeth were included. Class I cavity preparations were cut on occlusal surfaces. After crown separation, a flat dentinal surface was provided and RDT (remaining dentinal thickness) was adjusted at 0.5 mm and 1.5 mm in half of the samples respectively. Then, cavities were treated in three groups with experimental DBAs: Group 1: Scotch Bond Multipurpose, Group 2: Excite, Group 3: AdheSE. Blue inlay wax sealed the cavities. Crowns were immersed in culture medium for 24 hours and the cytotoxicity of the resultant toxic medium was measured both quantitatively with MTT assay and qualitatively with an inverted light microscope in 4 serial dilutions. Data were analyzed with ANOVA and Tukey’s test at 95% significance level.Results: Although morphologic cell changes were seen from mild to severe in serial dilutions, but MTT assay determined that only in neat dilution, cell changes were significantly different from controls. Besides, no significant differences were found between the three experimental DBAs regarding cytotoxic effect on L929 cell line.Conclusion: Considering the limitations of an in vitro study, if the RDT is less than 0.5 mm in Vivo, regardless of the type of DBA, destructive cellular changes in pulp tissue can be expected.