Objective To explore the mechanism of melatonin regulating Th1 / Th2 immune balance via targeting nuclear factor ( NF)-κB signaling pathway in mice with gastric cancer. Methods Gastric cancer cells ( 50 μg, approximately 1×10⁶ cells) were injected subcutaneously into the left forelimb of mice. After 7 days of modeling, tumor formation was felt, indicating that the model of transplanted gastric cancer tumor was successfully established. Ten healthy mice were used as the control group, whereas successfully modeled mice were divided into model and model+melatonin groups (n= 10 mice per group). The model+melatonin group was subcutaneously injected daily with melatonin (10 mg / kg, 1 time / d, 21 d), whereas the other groups were injected with the same amount of normal saline. Tumor volumes and masses were measured using a vernier caliper and balance, respectively. Proliferation and apoptosis of gastric cancer cells in tumor tissues were assessed by immunohistochemical detection of Ki67 and Bcl2. Levels of interferon ( IFN)-γ and interleukin ( IL)-4 in peripheral blood were detected by ELISA. Ratios of Th1 to Th2 cells in peripheral blood were detected by flow cytometry. Levels of NF-κB mRNA and protein were detected by qPCR and Western blot, respectively, in tumor tissues and lymphocytes. Results Contrast with the control group, the tumor volumes and masses, Ki67 and Bcl2 staining intensities, IL-4 levels, percentage of Th2 cells, NF-κB mRNA and protein expression levels were significantly increased (P<0. 05), and the IFN-γ levels, the percentage of Th1 cells and Th1 / Th2 ratio were significantly decreased in model and model+melatonin groups (P<0. 05). Contrast with model group, tumor volumes and masses, Ki67 and Bcl2 staining intensities, IL-4 levels, percentage of Th2 cells, NF-κB mRNA and protein expression levels were significantly decreased (P<0. 05) and the IFN-γ levels, the percentage of Th1 cells and Th1 / Th2 ratio were significantly increased in the model+melatonin group (P< 0. 05). Conclusions Melatonin may inhibit tumor growth in mouse models of gastric cancer and regulate the balance of Th1 / Th2 by regulating the NF-κB pathway.