Immune checkpoint blockade is an optional and effective therapy for bladder cancer. The present study was aimed to investigate the expression of programmed death ligand-2 (PD-L2) in patients with bladder cancer. Paraffin-embedded tissues of 92 patients with bladder cancer were obtained. Then immunohistochemistry of PD-L2 was performed. The expression intensity of PD-L2 was defined with score 0, 1, 2 and 3, in compliance with negative, weak, moderate, and strong, respectively. The association of PD-L2 expression with clinical characteristics was analyzed. A P < 0.05 was considered as significantly different. By defining the expression intensity of PD-L2 with score 0 to 3, 73.9% of patients (68/92) had a positive expression of PD-L2, and 43.5% (40/92) had a mediate or strong expression. Furthermore, high expression PD-L2 (mediate or strong expression) was more common among patients ≤ 70 y (P = 0.038) and those with smoke history (P = 0.045). The univariate Kaplan-Meier analysis indicated that high expression of PD-L2 was associated with both shorter overall survival (OS) (78.3 vs. 60.3 months; P = 0.037) and shorter disease-free survival (44.3 vs. 22.5 months; P = 0.004). The multivariate COX regression model showed that high expression of PD-L2 was a poor factor of disease-free survival (hazard ratio = 0.537, 95%CI 0.322-0.898; P = 0.018), but not OS (hazard ratio = 0.565, 95%CI 0.253-1.262; P = 0.164). Bladder cancer had a high expression of PD-L2. And high expression of PD-L2 may indicate worse prognosis. It may be a potential immunotherapeutic target of immune checkpoint blockade for bladder cancer. Copyright © 2020 Elsevier Inc. All rights reserved.