<p>SLC27A1 (solute carrier family 27 member 1), which encodes fatty acid transport protein 1 (FATP1), has been implicated in the development and progression of various cancers. However, its role in colorectal cancer (CRC) remains to be fully elucidated. This study combined a series of bioinformatics analyses and biological experiments to characterize the comprehensive function of SLC27A1 in CRC. The expression of SLC27A1 in CRC was analyzed using multiple databases. Bioinformatics methods were employed to explore the associations between SLC27A1 expression levels in CRC and its clinical significance, as well as immune infiltration. The function and potential mechanism of SLC27A1 in CRC were investigated through Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis, gene set enrichment analysis (GSEA), and in vitro experiments. SLC27A1 was significantly upregulated in CRC. Its high expression correlated with adverse clinicopathological features and poor prognosis in CRC patients. SLC27A1 expression was closely linked to immune cell infiltration in CRC. GSEA analysis revealed that SLC27A1 is involved in multiple tumor-associated signaling pathways and negatively correlates with the tricarboxylic acid cycle and pyruvate metabolism. In vitro experiments demonstrated that SLC27A1 knockdown inhibited CRC cell proliferation and migration. In contrast, SLC27A1 overexpression promoted glucose uptake and lactate accumulation in CRC cells. Further analysis showed that SLC27A1 was significantly associated with multiple lactylation-related genes. The results of this study demonstrate that high expression of SLC27A1 is closely associated with poor prognosis, protein lactylation, and immune cell infiltration in CRC. Knockdown of SLC27A1 can suppress the proliferation and migration abilities of CRC cells. Therefore, SLC27A1 may serve as a promising independent prognostic biomarker and a potential therapeutic target.</p>