Despite significant evidence that the expression of several microRNA’s (miRNA) impacts on disease activity in patients with ulcerative colitis (UC), it remains unknown if the more severe disease phenotype seen in pediatric-onset UC can be explained by altered miRNA expression. In this study, we aimed to assess the relationship between miRNA expression, age, and disease severity in pediatric and adult patients with UC. Using RT-qPCR, we analyzed the expression of miR-21, miR-31, miR-126, miR-142, and miR-155 in paraffin-embedded rectum biopsies from 30 pediatric and 30 adult-onset UC patients, and found that lesions from adult patients had significantly higher expression levels of miR-21 compared to pediatric patients and that the expression levels of miR-31 (all patients) and miR-155 (pediatric patients only) correlated inversely with histological assessed disease severity.