Many studies suggested that cytokines interleukin (IL)-29, IL-32, and tumor necrosis factor alpha (TNF-alpha) are implicated in the pathogenesis of malignancies. The purpose of this study was to determine the clinical significance of the serum levels of IL-29, IL-32, and TNF-alpha in gastric cancer (GC) patients. Fifty-eight GC patients and 20 age- and sex-matched healthy controls were enrolled into this study. The median age at diagnosis was 59.5 years (range 32-82 years). Tumor localization of the majority of the patients was antrum (n = 42, 72.4 %), and tumor histopathology of the majority of the patients was diffuse (n = 43, 74.1 %). The majority of the patients had stage IV disease (n = 41, 70.7 %). Thirty-six (62.1 %) patients had lymph node involvement. The median follow-up time was 66 months (range 1 to 97.2 months). The baseline serum IL-29 concentrations were not different between patients and controls (p = 0.627). The baseline serum IL-32 and TNF-alpha concentrations of the GC patients were significantly higher (for IL-32, p = 0.014; for TNF-alpha, p = 0.001). Gender, localization, histopathology, tumor, and lymph node involvement were not found to be correlated with serum IL-29, IL-32, and TNF-alpha concentrations (p > 0.05). Patients without metastasis (p = 0.01) and patients who responded to chemotherapy (p = 0.04) had higher serum IL-29 concentrations. Patients older than 60 years had higher serum IL-32 (p = 0.002). Serum IL-29, IL-32, and TNF-alpha levels were not associated with outcome (p = 0.30, p = 0.51, and p = 0.41, respectively). In conclusion, serum levels of IL-32 and TNF-alpha may be diagnostic markers, and serum IL-29 levels may be associated with good prognosis in patients with GC.