Background: Combination therapy of gemcitabine (GEM) and platinum agents and immune-checkpoint inhibitors are standard regimens for advanced urothelial carcinoma (UC), and optimum selection is important to improve the prognosis. Human antigen R (HuR) increases the protein expression of the enzyme deoxycytidine kinase, which is involved in metabolizing the prodrug GEM into its active metabolites. HuR expression is a predictor for the response to gemcitabine-based chemotherapy in a variety of cancers. This study evaluated the relationship between combination pattern of serum GEM level and HuR expression in cancer cells and anti-cancer effects of GEM-based regimens in patients advanced UC. Methods: Twenty-seven advanced UC patients (bladder = 8 and upper urinary tract = 19) were treated with a combination of GEM and platinum agents (cisplatin = 15 and carboplatin = 12) as first-line chemotherapy in Nagasaki University Hospital. Serum samples were collected 30 min after GEM injection. HuR expression was examined by immunohistochemical methods. Results: Positive expression of HuR was detected in 17 (63.0%) patients, and the mean/SD of serum GEM level was 14.9/5.1 μg/mL. C-HuR expression was not associated with anti-cancer effect (P = 0.066). Serum GEM level in the progressive disease (PD) group (10.4/4.7 μg/mL) was lower than that in groups with CR + PR (16.2/3.7) or SD (16.3/6.1); however, these differences were not significant. For the anti-cancer effect of the combination of serum GEM level and HuR expression, all 3 patients with low GEM levels (under median) and negative HuR expression showed PD. In contrast, among 7 patients with high GEM levels and positive C-HuR expression, no patients showed PD, and relationship between their combination pattern and anti-cancer effect was significant (P = 0.010). Conclusions: Serum GEM level or HuR expression was not associated with anti-cancer effects; however, their combination pattern was significantly associated. Although we examined a small number of patients, our results are useful for determining treatment strategies in patients with advanced UC.