Background Serum anti-proteinase 3 anti-neutrophil cytoplasmic antibody (PR3-ANCA) is a disease-specific antibody against granulomatosis with polyangiitis. Some patients with ulcerative colitis (UC) have positive results for PR3-ANCA but the clinical significance of PR3-ANCA is not clear. Thus, we conducted a multicenter, retrospective, observational study to elucidate the clinical significance of PR3-ANCA in UC.Methods In total, 150 patients with UC underwent colonoscopy and serum PR3-ANCA measurements. Activity was evaluated using the Mayo Endoscopic Subscore (MES), and the relationship between activity and PR3-ANCA was analyzed.Results Twenty-six of the 150 patients who met the exclusion criteria were eliminated; 124 patients were included. A positive correlation was observed between MES and PR3-ANCA (r = 0.42; p < 0.001). Receiver-operating characteristic analysis showed that the cut-off value for calculations was 4.1 U/mL. Of 108 patients with active-phase UC, 58 (53.7%) were positive for PR3-ANCA. Furthermore, patients for whom steroid therapy was ineffective had a significantly higher rate of being PR3-ANCA positive (p = 0.045). Of 21 patients who reached clinical remission with PR3-ANCA-positive active-phase UC, 13 had an MES ≥ 1 and 8 patients had an MES 0. For MES 0 cases, the reduction of PR3-ANCA levels was significant (p = 0.012) when comparing the active-phase and clinical remission. All cases with MES 0 were negative for PR3-ANCA. Only 4 of 13 cases with MES ≥ 1 were negative for PR3-ANCA.Conclusions Approximately half of UC cases are PR3-ANCA-positive during the acute phase, indicating that PR3-ANCA is a potential marker of disease activity that predicts steroid therapy failure.Trial registration This study was registered in the UMIN Clinical Trials Registry System (000039174). Retrospectively registered on 16th Jan. 2020.