Deoxynivalenol (DON) is one of the most commonly occurring trichothecenes, produced mainly by Fusarium graminearum. The project aimed to provide data on levels of total DON and de-epoxy deoxynivalenol (DOM-1) in human urine samples collected from different population groups (children, adolescents, adults, elderly, vegetarians, pregnant women) in Italy, Norway and the United Kingdom (UK) as analysed by liquid chromatography–mass spectrometry (LC-MS). Morning urine samples were collected over two consecutive days from 635 volunteers and associated food consumption was recorded on the same days. Levels of DON did not significantly differ between day 1 and day 2 urine samples. DON was detected in 99 %, 93 % and 76 % of the urine samples from Norway, UK and Italy, respectively. The median total DON concentrations were similar between population groups in Italy and Norway, but were approximately 3-fold higher in the sampled UK population. In Norway and the UK, levels of DON were roughly 2.5-fold higher in children compared with adults. For DOM-1, 12 % of Norwegian and 1.5 % of Italian urine samples were positive, but DOM-1 was not detected in any sample from the UK. This difference may be explained by differences across analytical sites in the limit of quantification (LoQ). Associations between food consumption and urinary DON levels were assessed by ordered logistic regression models. In Italy, intakes of pasta and pasta-like products were significantly associated with higher levels of total DON after correction for creatinine on both days. In Norway, intakes of breakfast cereals and snacks (day 1) and bread and bread-like foods (day 1 and 2) were significantly associated with a higher level of total DON adjusted for creatinine. In the UK, biscuit intakes on day 1 were significantly associated with a higher level of the toxin.