Aim The purpose of this study was to investigate the effects of dentine bonding agents on the expression of HO-1 in human pulp cells. Methodology Set specimens from Clearfil SE Bond (CB), Prime & Bond 2.1 (PB), and Single Bond (SB) were eluted with culture medium. Cytotoxicity and Western blot assays were used to investigate the effects of human primary pulp cells exposed to dentine bonding agents. Results Our data showed that CB, PB, and SB were cytotoxic to pulp cells in a concentration-dependent manner (p<0.05). The exposure of quiescent pulp cells to CB, PB, and SB resulted in the induction of HO-1 protein expression in a time-dependent manner (p<0.05). The influence of the cytotoxicity and HO-1 expression depended on the materials tested. Conclusion Taken together, HO-1 expression might be one signal transduction pathway linked to the induction of stress-inducible protein by dentine bonding agents.