Abstract Background There is no established diagnostic biomarker for ulcerative colitis (UC). Although we recently reported an anti-αvβ6 integrin antibody to diagnose UC, there is no large-scale validation study on this antibody to diagnose UC. This study aimed to validate the diagnostic value of anti-αvβ6 integrin antibody for UC in a nation-wide, multicenter cohort (UCV6-Dx Study). Methods We enrolled 1241 UC patients, 798 Crohn’s disease (CD) patients, and 207 other gastrointestinal disease patients from 28 Japanese high-volume referral centers. Anti-Integrin αvβ6 ELISA Kit (MEDICAL & BIOLOGICAL LABORATORIES CO., LTD., Tokyo, Japan) was used to measure the titer of anti-αvβ6 integrin antibody. The cut-off value was 1.64 U/mL (the mean +3SD of 83 serum samples from healthy volunteers determined by the manufacturer). The primary outcome was the diagnostic value of the anti-αvβ6 integrin antibody. Secondary outcomes were factors associated with false negative results in UC patients, factors associated with false positive results in non-UC patients, and the optimal cut-off values using receiver operating characteristic (ROC) analysis. Results The diagnostic sensitivity of the anti-αvβ6 integrin antibody in UC was 87.7% and the specificities were 82.0% in CD and 87.4% in other gastrointestinal diseases. Multivariate logistic regression analysis showed that false negative results in UC patients were associated with age at the time of sample collection odds ratio (OR) 1.0242, p=0.034, 95% confidence interval (CI) 1.0018-1.0470, smoker (OR 2.5504, p=0.002, 95% CI 1.4216-4.5755), partial Mayo score (OR 0.8623, p=0.038, 95% CI 0.7495-0.9921), and advanced therapies (OR 0.5875, p=0.019, 95% CI 0.3765-0.9167) and false positive results in CD patients with disease location L2 (OR 2.6858, p=0.013, 95% CI 1.2323-5.8533). No factor was associated with false positive results in patients with other intestinal diseases. ROC analysis revealed that the optimal cut-off values estimated by the Youden method were 2.07 U/mL area under the curve (AUC) 0.9003, sensitivity 85%, specificity 87%, and 1.78 U/mL (AUC 0.9206, sensitivity 87%, specificity 89%) for discriminating UC from CD and other gastrointestinal diseases, respectively. Conclusion The usefulness of anti-αvβ6 integrin antibody for diagnosing UC was validated in the Japanese large-scale, nation-wide multicenter study.