To evaluate the invitro biocompatibility of iRoot FS (Innovative BioCeramix Inc., Vancouver, BC, Canada) and to compare its performance with those of iRoot BP Plus (Innovative BioCeramix Inc.) and ProRoot mineral trioxide aggregate (MTA; Dentsply Tulsa Dental, Tulsa, OK, USA). MC3T3-E1 cells were cultured for 1, 2 and 3days in various dilutions of iRoot FS, iRoot BP Plus and MTA extracts after 7days of setting to assess cell viability using a cell counting kit-8 (Dojindo, Kumamoto, Japan). The cell apoptosis induced by the set material extracts was evaluated through annexin V-propidium iodide flow cytometry. Changes in the cytoskeletal organization and stress fibres were observed through immunofluorescence by labelling the fibrous actin and nuclei of the cells. Cell attachment was observed under a scanning electron microscope after the MC3T3-E1 cells were cultured on the surface of a material disc set for 1day. The data were analysed using one-way anova. iRoot FS extracts induced higher cell viability than the control extracts (P<0.05) at levels comparable to those of iRoot BP Plus and MTA. Compared with the control group, iRoot FS did not promote cell apoptosis. Stretched stress fibres and cytoskeletons were detected in the cells treated with iRoot FS extracts. Scanning electron microscopy revealed that the MC3T3-E1 cells attached to iRoot FS appeared flatter and exhibited better stretch than those attached to the other extracts. iRoot FS displayed invitro biocompatibility with MC3T3-E1 cells by promoting cellular proliferation and attachment without causing cell apoptosis.