Abstract Background Assessment of the disease activity in inflammatory bowel disease (IBD) is essential for adequate treatment management and reliable noninvasive biomarkers for verification of mucosal healing are still needed. MicroRNAs (miRNAs) are differentially expressed in IBD and cancer. We aimed to evaluate the potential of circulating and fecal miRNAs as diagnostic biomarkers for IBD. Methods In this proof-of-principle study we used 2 independent patient cohorts. Testing cohort (n = 96) included serum and fecal samples from controls (n = 35) and IBD patients (n = 61) including 43 patients with Crohn′s disease (CD), 18 with ulcerative colitis (UC) with an active disease (n = 38), or in remission (n = 23). Validation cohort included fecal samples from patients with calprotectin/endoscopy-confirmed active disease (n = 30) or in remission (n = 15). Target-based approach (miR-16, miR-21, miR-155, and miR-223) has been used to evaluate miRNA expression. Results Sera samples from IBD patients showed higher level of miR-16, miR-21, and miR-223, but not miR-155, compared to controls and was higher in CD than in UC patients. Much stronger miRNA expression changes were observed in feces from IBD patients for all studied miRNAs with highest expression of miR-155 and miR-223 in testing and validation cohorts. MiRNA expression correlated with clinical remission, however, only fecal but not circulating miRNAs, correlated with surrogate parameters such as fecal calprotectin or C-reactive protein. Conclusions Our data provide a novel evidence for differential expression level of fecal miRNAs in IBD. We demonstrate that miRNAs in feces correlate with disease activity and may be considered as potential tool for the further biomarker research in IBD. 10.1093/ibd/izy046_video1 izy046.video1 5794822319001