The purpose of this study was to assess the effects of iRoot SP root canal sealer (Innovative BioCreamix Inc, Vancouver, Canada) on the expression of mineralization-related genes in human MG63 osteoblast-like cells.Specimens (5 mm in diameter and 2 mm in height) of iRoot SP and AH Plus (Dentsply DeTrey, Konstanz, Germany) were extracted from a 5-mL culture medium. The MG63 cells were exposed to various dilutions (1/1, 1/2, and 1/4) of the extracts. The 3,(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide MTT assay was used for assessing the dental materials’ nonspecific cytotoxicity. The expression of mineralization-related genes, including collagen type I (COL I), osteocalcin (OCN), bone sialoprotein (BSP) and osteopontin (OPN), was detected on days 1, 3, and 6 by a real-time polymerase chain reaction. An enzyme-linked immunosorbent assay experiment was used for evaluating COL I and BSP protein changes. The data were analyzed with one-way analysis of variance and Tukey tests.In the MTT assay, the undiluted extracts of iRoot SP were noncytotoxic, whereas the undiluted extracts of AH Plus were rated as slightly cytotoxic. iRoot SP up-regulated COL I, OCN, and BSP messenger RNA expression after 3 and 6 days. In the enzyme-linked immunosorbent assay experiment, iRoot SP increased COL I and BSP protein levels compared with AH Plus and the control group on day 6.In the presence of iRoot SP, MG63 cells can produce more mineralized matrix gene and protein expression. Based on these results, iRoot SP can be considered as a favorable material for cell-material interaction.