A diagnosis of idiopathic inflammatory bowel disease requires synthesis of clinical, radiographic, endoscopic, surgical, and histologic data. While most cases of inflammatory bowel disease can be specifically classified as either ulcerative colitis or Crohns disease, 5-10% of patients have equivocal features placing them into the indeterminate colitis category. This study examines whether microRNA biomarkers assist in the classification of classically diagnosed indeterminate inflammatory bowel disease. Fresh frozen colonic mucosa from the distal-most part of the colectomy from 53 patients was used (16 indeterminate colitis, 14 Crohns disease, 12 ulcerative colitis, and 11 diverticular disease controls). Total RNA extraction and quantitative reverse-transcription-PCR was performed using five pairs of microRNA primers (miR-19b, miR-23b, miR-106a, miR-191, and miR-629). Analysis of variance was performed assessing differences among the groups. A significant difference in expressions of miR-19b, miR-106a, and miR-629 was detected between ulcerative colitis and Crohns disease groups (P<0.05). The average expression level of all five microRNAs was statistically different between indeterminate colitis and Crohns disease groups (P<0.05); no significant difference was present between indeterminate and ulcerative colitis groups. Among the 16 indeterminate colitis patients, 15 showed ulcerative colitis-like and one Crohns disease-like microRNA pattern. MicroRNA expression patterns in indeterminate colitis are far more similar to those of ulcerative colitis than Crohns disease. MicroRNA expression patterns of indeterminate colitis provide molecular evidence indicating that most cases are probably ulcerative colitis-similar to the data from long-term clinical follow-up studies. Validation of microRNA results by additional long-term outcome data is needed, but the data presented show promise for improved classification of indeterminate inflammatory bowel disease.