Background: Constipation is prevalent in pediatric cystic fibrosis (CF) patients and colonic motility has not been studied in this population. In this study, we aimed to evaluate the total and segmental colonic transit time in children and adolescents with CF based on the presence of constipation and radiological fecal impaction. Methods: In this case series, all patients aged 3 to 20 years of a CF reference center were invited to participate. CF-associated constipation was diagnosed based on the European Society for Paediatric Gastroenterology Hepatology and Nutrition criteria. Total and segmental colonic transit time was determined using radiopaque markers. Fecal impaction on plain abdominal radiography was assessed based on the Barr score. Results: Of the 43 eligible patients, 34 (79%) agreed to participate. Constipation was found in 44.1% of children and adolescents, predominantly in girls. The total colonic transit time (medians of 42 and 24 hours, respectively, P ¼ 0.028) and the segmental right colon transit time (medians of 8 and 2 hours, respectively, P ¼ 0.012) were significantly longer in CF-associated constipation group than in the group of patients without constipation. The frequency of radiological fecal impaction was similar in patients with (50.0%) and without (64.2%) CF-associated constipation (P ¼ 0.70). There was no relationship between radiological fecal impaction and the total and segmental colonic transit time. Conclusions: Children and adolescents with CF-associated constipation had a longer total and segmental right colon transit time. Colonic transit time was similar in patients with and without radiological fecal impaction.