Methacrylate monomers that are found to leach from cured resin-based dental materials induce biological effects in vitro. The underlying mechanisms have not been fully elucidated although involvement of increased cellular reactive oxygen species (ROS) and DNA-damage has been suggested. In this in vitro study we have elucidated the impact of a commonly used methacrylate monomer, HEMA, on the level and oxidation state of cellular glutathione, intracellular ROS level, as well as the formation of complex between HEMA and glutathione. HEMA exposure rapidly led to increased level of ROS and reduced level of GSH (reduced form of glutathione). Antioxidants effectively counteracted the ROS increase, but had no effect on the GSH depletion. No change in glutathione-disulphide (GSSG; oxidized form of glutathione) concentration was detected in the HEMA treated cells, showing that oxidation of glutathione was not responsible for the reduced GSH concentration. Further we demonstrated spontaneous formation of a complex between HEMA and GSH. In conclusion, we showed that exposure to HEMA led to drop in cellular glutathione level probably caused by complex formation with HEMA. A similar covalent binding of HEMA to macromolecules combined with increased level of cellular ROS due to lower levels of GSH is suggested to be important factors triggering the toxic response.