Natural compounds have generated great interest as alternative treatments of diseases like cancer. Here, we investigated the anti-tumor mechanism of one such compound, Agrocybe aegerita polysaccharide, by assessing expression of tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) in rat esophageal carcinoma (EC). EC was induced in healthy Wistar rats by methyl-n-amyl nitrosamine. Subsequently, rats were administered cancer treatment daily for 4 weeks, as follows: the normal control group (the only group not treated with methyl-n-amyl nitrosamine) and model group received only distilled water; the chemotherapy group received tegafur treatment; and the combination group received tegafur combined with A. aegerita polysaccharide. In normal and combination groups, body weight increased gradually after each week of treatment (P < 0.05), while body weights did not change in model and chemotherapy groups. Using enzyme linked immunosorbent assay, we found serum TNF-α was lower in the combination group (31.56 ± 7.20 pg/L) than either the model (46.24 ± 8.52 pg/L) or chemotherapy (52.39 ± 9.16 pg/L) group, and, while higher, was more similar to the normal controls (25.08 ± 2.93 pg/L; P < 0.05), a finding that was confirmed by the immunohistochemistry of esophageal samples. In contrast, serum IFN-γ was higher in the combination group (97.20 ± 10.92 pg/L) than in either the model (76.11 ± 11.92 pg/L) or chemotherapy (76.04 ± 9.85 pg/L) group, but lower than in the normal group (117.56 ± 10.88; P < 0.05), also confirmed by immunohistochemistry. Therefore, Agrocybe aegerita polysaccharide, when combined with chemotherapy, can regulate immune function in EC, potentially by modulating cytokine activity, specifically downregulation of TNF-α and upregulation of IFN-γ.