Although the induction of heme oxygenase-1 (HO-1) by hydrogen peroxide (H2O2) has been reported, the HO-1 and odontoblastic differentiation-inducing effects against H2O2 have not been clarified in human pulp cells. In this study, we investigated whether HO-1 is involved in the protective mechanisms against the cytotoxic effects of H2O2 by using a cell viability assay, and we examined the production of dentin sialophosphoprotein (DSPP) and other mineralization markers by using reverse transcriptase-polymerase chain reaction in human pulp cells. H2O2 decreased cell viability but increased HO-1 and DSPP expression in a concentration- and time-dependent manner. Antioxidants and inhibitors of HO-1, phosphatidylinositol-3'-kinase, extracellular signal-regulated kinase, and p38 mitogen-activated protein kinase blocked H2O2-induced cytotoxicity and the expression of HO-1 and DSPP mRNA in pulp cells. These data suggest that the induction of HO-1 by H2O2 in pulp cells plays a protective role against the cytotoxic effects of H2O2 and stimulates DSPP expression, resulting in premature odontoblast differentiation through pathways that involve phosphatidylinositol-3'-kinase, p38, and extracellular signal-regulated kinase.