Introduction: The aim of this study was to compare the cytotoxicity of Mineral Trioxide Aggregate (MTA) and a New Endodontic Cement (NEC) on L929 mouse fibroblasts. Materials and Methods: Different dilutions (Neat, 1/2, 1/10, 1/100) of fresh and set materials placed adjacent flasks of L929 in DMEM medium. Cellular viability was assessed using MTT assay in three time intervals (24, 48, and 72 h after mixing). Differences in mean cell viability values between materials were assessed by using the One-way ANOVA and Bonferoni post-test. Optical microscopic analysis of morphology of the untreated control and the cement-treated cell cultures were carried out in all experimental periods. Results: It was indicated that there was not a significant difference in cytotoxicity among the materials of test and between them and the control group. However, there was a statistically significant difference between different time intervals within each group (P< 0.05) and between different concentration of test materials (P<0.05). In all samples, set materials showed better viability than fresh ones. Conclusion: According to results of this study, NEC and MTA have similar cytotoxic effect on L929 cell culture.