Selenium (Se) is a trace element essential to cellular functions in humans, categorized into ‘organic’ and ‘inorganic’ Se chemical forms. Epidemiological studies in several countries found an association between increased Se intake and reduced risk of cancer, but clinical trials that followed up on this association have found controversial outcomes. Many in vitro studies have focused on the role of Se in DNA damage to better to understand how Se may be involved in cancer prevention. However, the effect of Se in mutagenesis, an important aspect of carcinogenesis, is less well-understood, due to differences observed depending on concentrations, chemical form and treatment schedules. To the best of our knowledge, there are no human in vitro mutation studies investigating Se. The research presented here focused on adapting an established mutagenesis assay to look at the impact of selenium on mutation frequency and anchor this to information on DNA damage and phosphorylation changes. After establishing a reliable TK6 gene mutation assay, it was found that pre-treatment with organic Se reduced mutation frequency in chemical mutagen and low-dose radiation systems while inorganic Se did not. ROS levels were also reduced by SeY but not selenite, with some differences in antioxidant activity and protein expression also observed. DNA damage pathways were investigated using PCR; these showed differential profiles observed between Se treatments shortly after mutagen exposure; SeY showed significant inhibition of the p53 pathway, while selenite showed early upregulation of apoptotic genes, consistent with MF and toxicity data. Phosphorylation changes supported the findings of the DNA damage data, with inhibition of phosphorylation of p53 and Chk2 observed with SeY and enhanced CDDP-signalling with selenite treatment. This research contributes to an expanding body of knowledge on the role of Se and dissection of its form-specific effects on mutagenesis and the potential implication in carcinogenesis.