Abstract: Colorectal cancer (CRC) is a highly prevalent form of cancer on a global scale. In 2022, global statistics indicated that more than 1.9 million new colorectal cancer cases and 930 000 deaths occurred worldwide. Its incidence and mortality rates rank third and second among malignant tumours, respectively. The development of colorectal cancer is the result of multiple complex molecular mechanisms, given the highly heterogeneous and complex nature of the disease. Long non-coding RNA (LncRNA) is a class of functional RNA molecules that are characterised by a length of more than 200 nucleotides and an absence of protein-coding potential. LncRNA is extensively present in the human genome and plays a pivotal role in cancer development by regulating the proliferation, invasion, metastasis, and metabolic reprogramming of tumour cells. Long intergenic non-coding RNA 1063 (LINC01063) is an LncRNA that is highly expressed in colorectal cancer, but its mechanism of action has not yet been fully elucidated. Single-cell metabolomics has been demonstrated to reveal metabolic and phenotypic diversity among cells, thereby providing a more comprehensive understanding of cellular phenotypes when compared to genomics and transcriptomics. In this study, the effect of LINC01063 on the metabolism of DLD-1 colorectal cancer cells was investigated using intact living-cell electro-launching ionization mass spectrometry. Single-cell metabolomics was performed on 1 397 and 1 946 DLD-1 cells with LINC01063 expression interfered with. A total of 1254 metabolite-associated ions were detected in the two cell types, with 1 163 and 960 ions detected in the two cell types, respectively. The dimensionality reduction analysis of single-cell metabolic data enabled the differentiation of DLD-1 cells before and after LINC01063 gene interference, and the metabolic pathways obtained from population cells were compared with those from singe cells for analysis. Among them, 15 pathways were identified in both analysis methods, and the single-cell analysis additionally identified 12 unique metabolic pathways, such as glycerophospholipid metabolism and nicotinate and nicotinamide metabolism. This finding indicated that single-cell metabolic analysis has the potential to unveil metabolic features that are not discernible in population cells. The investigation revealed that interference with the LINC01063 gene primarily affected amino acid and lipid metabolism pathways in DLD-1 cells, as determined by single-cell metabolomics. A combination of mass spectrometry-based single-cell metabolic technology and rigorous research methods was utilized to investigate metabolic changes in colorectal cancer cells before and after LINC01063 interference. The present study has utilized a comprehensive single-cell metabolomics analysis to elucidate the mechanism by which LINC01063 exerts its effect on colorectal cancer cells, and can provide a valuable reference for future research in this field.