The incidence of ulcerative colitis (UC) is on the rise also in Japan. Simultaneously, therapeutic options, including biologics and Janus kinase (JAK) inhibitors, have significantly expanded over the past decade. Although tofacitinib (TOF), one of JAK inhibitors, is a viable option for patients with moderate to severe UC, there is insufficient data to predict responsiveness of TOF treatment. The present study aimed to determine whether the infiltration of IL-17 A-positive mononuclear cells into the colonic mucosa can predict responsiveness to TOF treatment. Patients with UC who underwent TOF treatment were divided into responder and failure groups. Subsequently, we conducted a comparative analysis to identify differences in the infiltration of IL-17 A-positive cells into the colonic mucosa through immunohistochemical examination of colon biopsy samples. The proportion of IL-17 A positive mononuclear cells in colon biopsy samples was significantly higher in the failure group than among responders (38.2% vs. 21.2%). Consistent with this finding, our re-analysis of RNA sequence datasets available in the Gene Expression Omnibus (GEO) database suggested that TOF exerts a more pronounced influence on Th1 cells compared with IL-17-producing Th17 cells. In summary, an abundance of IL-17 A-positive mononuclear cells in the colonic mucosa has the potential to predict the responsiveness to TOF treatment.