Aim: Intestinal tuberculosis (ITB) and Crohn's disease (CD) have overlapping clinical, radiographic, endoscopic and histologic features, which makes the distinction between these two disease entities a great challenge in tuberculosis-endemic countries. The aim of the study was to investigate the value of in vitro interferon), release assay (T-SPOT.TB) in differentiating ITB from CD. Methods: From June 2008 to February 2010, a total of 93 consecutive patients with undetermined ITB or CD were prospectively recruited. Clinical, endoscopic, histologic and therapeutic responses were longitudinally monitored at follow-up evaluation until the final definite diagnosis has been reached. Results: After a median of 6 months' follow-up (interquartile range [IQR], 3.0 to 7.5 months), definitive diagnosis was achieved in 84 of the 93 patients (90%), with 19 having ITB and 65 having CD. On univariate analysis, a long duration of illness, chronic diarrhea, and anemia were significantly more common in CD (P<0.05). While night sweat, ascites, pulmonary lesions, circumferential ulcer on endoscopy, ileo-cecal valve involvement and epithelioid granulomas were significantly more common in ITB (P<0.05). On multivariate analysis, T-SPOT.TB (Hazard ratio 7.0, 95% confidence interval [CI] 1.9-25.7) was found to be a good predictor for ITB diagnosis. The sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) of T-SPOT.TB were 84.2%, 75.4%, 50.0%, and 94.2%, respectively. Conclusions: When differentiating ITB and CD in tuberculosis-endemic regions, T-SPOT.TB blood test may be a helpful and practical diagnostic tool for its high NPV to rule out ITB. Crown Copyright (C) 2012 Published by Elsevier B.V. on behalf of European Crohn's and Colitis Organisation. All rights reserved.