The DNA mismatch repair (MMR) system plays an important role in the initiation, diagnosis and treatments of colorectal cancer (CRC). Compared to CRC patients deficient in DNA MMR (dMMR), CRC patients proficient in DNA MMR (pMMR) have higher metastasis, short survival and poor response to chemotherapy and immunotherapy. It is well‑known that a high‑fat diet can cause CRC, and lipid metabolism is closely related to the development and metastasis of CRC. However, there have been few studies that address the difference in lipid metabolism between dMMR and pMMR CRC. Liquid chromatography‑tandem mass spectrometry (LC/MS) is an advanced technique that can perform the analysis of lipid metabolites and the roles of lipids present in low abundance in cell signaling and membrane stability. In the present study, we used the LC/MS technique to analyze the difference in the lipid metabolic profiles between dMMR cell lines (HCT116, DLD1, LoVo and HCT15) and pMMR cell lines (SW480, SW620, HT29 and NCM460). The results revealed that, among the 19 classes and 157 intact lipid species identified by the LC/MS analysis, the levels of most phospholipids were lower in dMMR cells than pMMR cells. Higher levels of phosphatidylcholine (PC; 16:0/18:1) and phosphatidic acid (PA; 18:0/18:0) were observed in pMMR cells than in dMMR cells. Furthermore, our results revealed that SCD1 and PLD1, the key enzymes involved in lipid metabolism associated with metastasis, are higher in pMMR cells than dMMR cells. To the best of our knowledge, we are the first to reveal that the levels of metastasis‑associated lipids and key enzymes in lipid metabolism were higher in the CRC patients with pMMR compared with the CRC patients with dMMR. This study identified potential anti‑metastatic targets in the therapy of patients with pMMR, and also personalized therapy for the patients with pMMR.