Background: In patients with advanced urothelial cancer (UC), a combined therapy of cisplatin (CDDP) and gemcitabine (GEM) is the most commonly used first (1st)-line systematic chemotherapy. Although no regimen for CDDP-resistant UC has been established, GEM-based regimens are often used in these patients. In other cancers, HuR status in cancer cells is closely associated with response to GEM. The aim of this study was to establish the predictive value of HuR expression for disease progression and survival in UC patients treated with GEM-based regimens as 1st or second (2nd)-line chemotherapy. Methods: Fifty patients with advanced UC were included in the study. As 1st-line chemotherapy, MVEC (methotrexate, vinblastine, epirubicin, and CDDP) and GC therapy were performed in 34 (68.0%) and 16 patients (32.0%), respectively. After progression, 45 patients (90.0%) were treated with combined GEM and paclitaxel (PTX) therapy, and 5 patients (10.0%) were treated with GEM monotherapy. Cytoplasmic and nuclear Human antigen R (HuR) expression was evaluated using immunohistochemical techniques. The relationships between HuR expression and local response and outcome were analyzed. Results: In 1st-line chemotherapy, no anti-cancer effects were associated with nuclear or cytoplasmic HuR expression. In 2nd-line chemotherapy, although nuclear HuR expression also had no significant relationship to anti-cancer effects, local tumor response was significantly better if there was positive cytoplasmic HuR expression (P = 0.002). Multivariate analyses revealed that cytoplasmic HuR expression was a significant predictive marker for longer overall survival (hazard ratio, 0.22; 95% confidential interval, 0.09–0.56; P = 0.001). There is no significant relationship between nuclear HuR expression and parameters of anti-cancer effects. Conclusions: Cytoplasmic HuR expression is a significant predictive marker of response to GEM-based chemotherapy in CDDP-resistant UC. Despite limitations of a small retrospective study, our results might have important information to discuss the treatment strategies and warrant further basic and clinical research.