Aim: The aim of the present study was to evaluate the cytotoxic effects of an epoxy resin sealer (AH Plus, Dentsply DeTrey, Konstanz, Germany) using a three-dimensional (3D) cell culture model. A conventional two-dimensional (2D) cell culture model was used as a reference technique for comparison. Materials and methods: Balb/c 3T3 fibroblasts were cultured in conventional two-dimensional cell culture and in rat tail collagen type I three-dimensional cell culture models. Then, cell cultures were incubated with elutes of freshly mixed AH Plus and with elutes of AH Plus after 7 days of mixing for 24 h. Cell viability was measured by the methyl-thiazol-diphenyltetrazolium assay. Data were statistically analysed by an independent t-test (P 0.05). Results: In all tested conditions, AH Plus exhibited cytotoxic effects; however, it was cell- and timedependent. In both conditions, AH Plus was highly cytotoxic in fresh conditions (P 0.05). Moreover, AH Plus showed high cytotoxic effects in the 2D cell culture model when compared to the 3D cell culture model (P 0.05). Conclusion: A 3D cell culture can be used as an alternative in vitro experimental model, able to provide reliable guidance on endodontic sealer toxicity under more closed conditions, related to the physiological scenario found in real-life 3D tissue microenvironments. Keywords: 3D cell culture, cytotoxicity, Epoxy Resin, root canal sealers