In animals deoxynivalenol (DON) causes vomiting, feed refusal, growth retardation, and affects the immune system. DON is a common contaminant of wheat, however, validated biomarker data to assess exposure at the individual level and therefore any associated health effects are lacking. The development of a highly robust assay for urinary DON involving immunoaffinity (IAC) clean-up and liquid chromatography (LC)-mass spectrometric (MS) detection has allowed the assessment of (1) DON exposure within UK individuals and (2) a wheat intake intervention on urinary DON levels. Twenty-five volunteers from the United Kingdom (aged 21-59 years) completed semi-weighed food diaries on days 1 and 2 (normal diet), and a morning urine sample was provided on day 3. On days 3-6 (intervention), individuals restricted major sources of wheat intake following dietary guidance. Diaries were completed on days 5 and 6, and a further morning urine was provided on day 7. Urinary DON was measured following IAC clean-up and analysis by LC-MS. Wheat-based food intake (mean 322 g/day, range: 131-542 g/day), was significantly (P<0.001) reduced during the intervention to 26 g/day (range: 0-159 g/day) indicating good compliance. DON was detected in all 25 urine samples taken on day 3 (geometric mean 7.2 ng DON/mg creatinine (95% confidence interval (CI) 4.9-10.5 ng/mg), but following the intervention there was a significant 11-fold reduction (P<0.001) to 0.6 ng per mg (95% CI 0.4-0.9 ng/mg). These data are unique in demonstrating human exposure to DON in the United Kingdom using a urinary biomarker. Furthermore, the study demonstrates that exposure can be markedly reduced by avoiding wheat in the diet. On the basis of urinary biomarker levels some individuals are predicted to exceed current recommended daily intakes of DON, and thus the health consequences of these exposures merit further investigation.