Background: Given the limitations of the use of common endodontic irrigants such as sodium hypochlorite and chlorhexidine (CHX), researchers are seeking out new irrigants with less complications. The purpose of this study was to compare the cytotoxicity of cetylpyridinium chloride (CPC) with sodium hypochlorite, CHX and Halita as an endodontic irrigant using MTT assay. Methods: In the present experimental study conducted from April 2016 to June 2018 in Tabriz University of Medical Sciences, cytotoxicity of CPC (0. 05%), CHX (0. 2%), sodium hypochlorite (2. 5%) and Halita solutions was examined on human gingival fibroblast cell lines according to the standard MTT assay protocol. The solutions were diluted at ratios of 1, 0. 1, 0. 01 and 0. 001. Thus, four concentrations of each solution were prepared and evaluated. Data were analyzed using descriptive statistical methods and paired t test, one-way ANOVA, repeated measures ANOVA, and post hoc tests. P value <0. 05 was considered significance level. Results: In the first 24 hours, the lowest cytotoxicity was observed for CHX (6. 19 ± 3. 10) and CPC (7. 08 ± 3. 04) at dilution of 0. 001 and the highest cytotoxicity was observed for Halita solution (25. 15 ± 7. 02) and sodium hypochlorite (22. 91 ± 7. 77) at dilution of 0. 01 (P < 0. 05). In total, the cytotoxicity of CPC at both concentrations and at all intervals was similar to CHX (P > 0. 05) and lower than two other solutions (P < 0. 05). At 24-hour interval, cytotoxicity of the solutions at both dilutions was lowest (P < 0. 05). At 48 and 72-hour intervals, the cytotoxicity of the solutions increased at both dilutions; however, there was no significant difference in mean cytotoxicity between 48-and 72-hour intervals (P > 0. 05). Conclusions: All solutions, particularly at commercial doses, had some levels of cytotoxicity depending on time and dose. The cytotoxicity of CPC 0. 05%, at all intervals and at the dilutions of 0. 01 and 0. 001, was similar to the cytotoxicity of CHX and lower than the cytotoxicity of sodium hypochlorite and Halita, and therefore CPC 0. 05% can be replaced with CHX in the presence of favorable antibacterial effects.