Objectives: The effect of dentin contacting materials on three-dimensional cultures of pulp-derived cells was evaluated in a dentin barrier test device using erbium-doped yttrium, aluminum, and garnet (Er:YAG) lasertreated dentin. Methods: The test materials (iBond®, G-Bond™, and Vitrebond™) were applied on lasertreated or untreated dentin discs. After 24 h of exposure with perfusion of the test chamber, cell survival was evaluated by enzyme activity and related to a nontoxic control material. The mean values of control tissues were set to represent 100% viability. Data were analyzed using Kruskal-Wallis and Mann-Whitney U test. Results: Vitrebond was the most toxic material for both laser-treated and untreated dentin. On untreated dentin, G-bond was cytotoxic to the pulp-derived cells (p < 0.05), and iBond was similar to the negative control group (p > 0.05). However, G-Bond and iBond were not cytotoxic when they were applied to Er:YAG lasertreated dentin (p > 0.05). Conclusion: Er:YAG laser treatment of dentin may protect the pulp cells from toxic substances of dentin contacting restorative materials; however, this effect is material related. Taking into consideration the limitations of this in vitro study, the Er:YAG laser treatment of dentin before restoration might be an option for decreasing the cytotoxic effects of the dental materials. Further research is required for clinical applications. © The Author(s) 2014.