Sulphur mustard (SM) is a blistering agent that has been used several times as a weapon during military conflict. Interest in this compound persists due to its ease of production and potential threat as an agent of warfare/terrorism. In addition, there are increasing reports of long-term health effects in individuals previously exposed to this compound, including an increased incidence of certain cancers. It is therefore important to elucidate the toxic mechanisms of SM and how the cell responds to any damage produced. This will allow for better healthcare planning in the event of an exposure and aid in the development of a therapeutic strategy, which is currently lacking. SM is a bifunctional alkylating agent, producing both DNA monoadducts and crosslinks, although the cellular response to these lesions is not well understood. This study aimed to investigate the DNA repair pathways employed by cells exposed to SM. It was found that DNA double strand breaks were generated after SM exposure and cells lacking the homologous recombination DNA repair pathway were more sensitive to the toxicity of SM than wild type cells. Finally, we demonstrate that chemical activation of the HR protein RAD51 offers cellular protection against SM toxicity and thus could be a novel target for therapeutic intervention.