Objective To detect the expression of myelodysplastic leukemia factor 1-interacting protein (MLF1IP), naked cuticle homolog protein 1 (NKD1), and Golgi phosphoprotein 3 (GOLPH3) in gastric cancer tissues, and to determine the relationship between them and the level of immune cell infiltration and drug sensitivity. Methods The research subjects were the cancer and adjacent tissues of 51 patients with gastric cancer who were treated at the First People's Hospital of Shangqiu City from January 2022 to January 2024. The expressions of GOLPH3, MLF1IP and NKD1 were detected by immunohistochemistry. Analyze the relationship between the expression of MLF1IP, NKD1 and GOLPH3 and the infiltration level of immune cells as well as the sensitivity to different drugs. Results Compared with adjacent tissues, the expression of GOLPH3, the level of macrophages, the expression of NKD1 and the expression of MLF1IP in gastric cancer tissues were higher, while the levels of CD8 ⁺T cells, B cells, CD4 ⁺T cells, neutrophils and dendritic cells were lower. The differences were statistically significant (P<0.05). The drug resistance of doxorubicin (ADM) and cisplatin (DDP) in gastric cancer tissues was higher than that of fluorouracil (5-FU) and mitomycin (MMC), and the difference was statistically significant (P<0.05). NKD1, MLF1IP and GOLPH3 were negatively correlated with CD8 ⁺T cells, neutrophils, B cells, CD4+T cells and dendritic cells, and positively correlated with macrophages. Compared with the negative expressions of NKD1 and MLF1IP, the positive expressions of NKD1 and MLF1IP have higher drug resistance and lower sensitivity for MMC, DDP, 5-FU and ADM. Compared with the negative expression of GOLPH3, the positive expression of GOLPH3 had higher sensitivity and lower drug resistance in ADM, 5-FU, DDP and MMC, and the difference was statistically significant (P<0.05). Conclusion MLF1IP, NKD1 and GOLPH3 are highly expressed in gastric cancer tissues, closely related to the sensitivity of chemotherapy drugs, and can inhibit the infiltration of immune cells.