In this paper, we report a study on the clinical relevance of prothymosin-α expression and its correlation with intratumoral Foxp3(+) and CD8(+) lymphocytes (Foxp3(+)TIL and CD8(+)TIL) in bladder cancer patients. We used immunohistochemical staining for prothymosin-α, Foxp3, and CD8 on 101 tumor specimens harvested by endoscopic resection. The results were correlated with clinicopathological variables and clinical outcome in bladder cancer patients, particularly in 73 patients with superficial disease, using the log-rank test and Cox proportional hazard model. Overall, of the tumors, 30 % were negative, 34 % showed nuclear, and 37 % showed cytoplasmic prothymosin-α expression. Foxp3(+)TILs were detected in 11 % of patients (nonnuclear vs. nuclear, p = 0.096). Patients with a history of urothelial carcinoma have a higher frequency of nonnuclear prothymosin-α expression than those without (p = 0.016, chi-square test). By univariate and multivariate analyses of cases with superficial disease, grade and stage were identified as independent predictors for recurrence-free survival (p = 0.016 and 0.016, respectively). Higher stage and nonnuclear prothymosin-α expression independently predict shorter progression-free survival (p = 0.006 and 0.043, respectively). The presence of Foxp3(+)TILs was significantly associated with disease progression by univariate analysis (p = 0.022), but not by multivariate analysis (p = 0.147). In vitro assays showed that J82 cells which express ectopically nuclear prothymosin-α exhibit higher growth rate and secrete less TGF-β1 than those with cytoplasmic expression or control cells. Altogether, prothymosin-α expression is a determinant of disease progression in superficial bladder cancer. Foxp3(+)TILs tend to be found more often in bladder cancer with nonnuclear prothymosin-α expression. Future study is required to unravel their interaction.