Background. Dientamoeba fragilis is commonly identified in children in primary care and is suspected to cause gastrointestinal disease. Objective. To determine the association between D. fragilis colonization and gastrointestinal symptoms in children. Methods. We performed a cross-sectional study with children who presented in primary care with gastrointestinal symptoms. The associations between D. fragilis colonization and specific symptoms were explored by means of logistic regression analyses. Asymptomatic siblings of these cases were invited as control subjects for a case-control analysis, where we explored the association between D. fragilis and gastrointestinal symptoms with conditional logistic regression analysis. Results. In the cross-sectional study, 107 children were included. Their median age was 9 years (interquartile range = 6-12) and 38 (35.5%) were boys. Colonization of D. fragilis was present in 59 children (55.1%). The absence of D. fragilis was associated with soft to watery stool [odds ratio (OR) = 0.29; 95% confidence interval (CI) = 0.10-0.85], chronic diarrhoea (OR = 0.42; 95% CI = 0.180.97) and fatigue (OR = 0.45; 95% CI = 0.20-0.99). The case-control analyses included 44 children in each group. Dientamoeba fragilis colonization was not observed more often in cases than in controls after adjustment for age and sex (OR = 1.02; 95% CI = 0.28-3.65). Conclusion. Dientamoeba fragilis is a common parasite in children with and without gastrointestinal symptoms. The anomalous finding of the association between the absence of D. fragilis with soft to watery stools, chronic diarrhoea and fatigue are inexplicable. Our study suggests that D. fragilis colonization does not increase the risk for gastrointestinal symptoms.