Gastric cancer is a major public health concern as the fourth most common cancer, and it is of particular relevance as the second most common cause of cancer death worldwide. We caparisoned the urinary nucleoside concentrations between the gastric patients and healthy volunteers that try to evaluate the diagnostic value in the gastric cancer. Urinary nucleosides from 49 gastric patients and 40 healthy volunteers were evaluated by highperformance liquid chromatography/electrospray ionization–tandem mass spectrometry (HPLC/ESI–MS/MS) under optimized conditions as determined in our previous study. The mean concentrations of 5 urinary nucleosides, cytidine, 3-methylcytidine (m3C), 1-methyladenosine (m1A), adenosine, and inosine, were found to be elevated in cancer patients, but only cytidine showed a significant elevation. Moreover, cytidine concentrations were significantly elevated by an average of 1.42-fold in patients with late stage (S3 + 4) disease. Combining the determined concentrations of preoperative serum alpha-fetoprotein (AFP, cutoff of 20 μg/l) or carbohydrate antigen 19-9 (CA19-9, cutoff of 37 U/ml) with the mean urinary cytidine concentration was shown to improve the diagnostic ratio (sensitivity) for gastric cancer from 16.3% (8/49 patients) to 38.8% (8 + 11/49 patients) or from 28.6% (14/49 patients) to 51.0% (14 + 11/49 patients), respectively. Urinary cytidine may be an important adjunct biomarker for gastric cancer.