Anti-proteinase 3 anti-neutrophil cytoplasmic antibodies (anti-PR3 ANCA) represent an established serologic marker of active granulomatosis with polyangiitis, but their role as a serologic marker in inflammatory bowel disease (IBD) remains uncertain. This study evaluates the presence of anti-PR3 ANCA and their validity as a serologic marker to aid in the diagnosis of IBD. Retrospectively, 142 serum samples obtained at early stages of the disease were analyzed with a new chemiluminiscent assay for the measurement of anti-PR3 ANCA. The results were correlated to the diagnosis, clinical, and therapeutic data, and ANCA and anti-Saccharomyces cerevisiae antibody (ASCA) measurements available from routine clinical practice. Anti-PR3 ANCA were significantly more prevalent (p < 0.0001) and their titers significantly higher (p < 0.0001) among ulcerative colitis compared with Crohn's disease patients. Receiver operating characteristic curve analysis performed with anti-PR3 ANCA titers to assess the diagnostic accuracy of the assay gave an area under the curve of 0.81 (95 % CI (0.76-0.89); p < 0.0001), with a cut-off titer of 11.8 chemiluminescent units displaying 52.1 % sensitivity and 97.3 % specificity for ulcerative colitis. Combining anti-PR3 ANCA positivity with IgA ASCA negativity as the diagnostic parameter demonstrated highest diagnostic utility, with a sensitivity and specificity of 47.5 % and 98.2 %, respectively. In our cohort, anti-PR3 ANCA was significantly more prevalent in ulcerative colitis than in Crohn's disease patients, which suggests a possible role of anti-PR3 ANCA as a serologic marker to aid in the diagnosis of IBD.