Objective To investigate the anti-tumor effect and immunological mechanism of lactobacillus casei in a rat model of gastric cancer. Methods Fifty male SD rats were randomly divided into five groups: control, model, and low, medium and high dose groups with 10 rats in each group. The model group and the three dose groups were treated with N-methyl-N-nitro-nitrosoguanidine (MNNG) to establish the gastric cancer model, and the control group was administered an aqueous alcohol solution. From week 28, the three dose groups were administered 4, 8, and 12 mL/ kg lactobacillus casei by gavage,respectively, and 10 mL/ kg sterile saline was administered to the model and control groups per day for 8 weeks. Indexes of the thymus and spleen were compared. Natural killer ( NK) cells ( TCRαβ⁺ CD161a⁺ ) and T lymphocyte subsets (CD4 ⁺ , CD8 ⁺ , and CD4 ⁺ / CD8 ⁺ ) in blood and tumor tissues were detected and compared. Relative protein expressions of Toll-like receptor-4 (TLR-4) and NF-κB p65 were compared. Results Pathological observations revealed that inflammatory cells infiltrated into the fore and gland stomach of the model group, keratinization of squamous epithelial cells occurred, and cancer cells were found under a light microscope. Inflammatory cells in the fore and gland stomach of the three dose groups were decreased, and the number of cancer cells was decreased with the most significant decrease in the middle dose group. Indexes of the thymus and spleen, TCRαβ⁺ CD161a⁺ , CD4 ⁺ , and CD4 ⁺ / CD8 ⁺ cells in peripheral blood, and TCRαβ⁺ CD161a⁺ and CD8 ⁺ cells in tumor tissues of the model group were significantly lower than those of the control group ( P < 0. 05). The above indicators of the three dose groups were significantly higher than those of the model group ( P <0. 05), and those of the middle dose group were significantly higher than those of low and high dose groups ( P <0. 05), but there were no significant differences between the low and high dose groups ( P >0. 05).CD4 ⁺ and CD4 ⁺ / CD8 ⁺ cells, relative protein expression of TLR-4 and NF-κB p65 in tumor tissues, and CD8 ⁺ cells in peripheral blood of the model group were significantly higher than those of the control group ( P < 0. 05). The above indicators of the three dose groups were significantly lower than those of the model group ( P < 0. 05), and those of the middle dose group were significantly lower than those of the low and high dose groups ( P < 0. 05), but there were no significant differences between the low and high dose groups ( P > 0. 05). Conclusions Lactobacillus casei modulate the activity of T lymphocytes and NK cells in tumor tissues and peripheral blood of gastric cancer model rats, and inhibit tumor growth and enhance immune regulation by downregulating protein expression of TLR-4 and NF-κB p65 and inhibiting the TLR-NF-κB signaling pathway. Among the groups, 8 mL/ kg Lactobacillus casei had the optimal effects.