AbstractObjectiveTo characterize alpha‐fetoprotein (AFP)‐producing gastric cancer (AFPGC) at the single‐cell level and to identify regulatory factors for AFP expression and malignancy.MethodsScRNA‐seq was performed on two tumors collected from patients with AFPGC. InferCNV and sub‐clustering were applied to identify typical AFPGC cells, followed by AddModuleScore, pathway enrichment, Pseudo‐time, and Scenic analyses. Data from a gastric cancer (GC) cohort were collected for conjoint analysis. The analytical results were verified by cell experiments and immunohistochemistry.ResultsAFPGC cells are similar to hepatocytes in transcriptome and transcriptional regulation, with kinetic malignancy‐related pathways, compared to the common malignant epithelium. In addition, compared to common GC cells, malignancy‐related pathways, such as epithelial‐mesenchymal transition (EMT) and angiogenesis, were upregulated in AFPGC. Mechanistically, Dickkopf‐1 (DKK1) was found to be associated with AFP expression and malignant phenotype upon combining our scRNA‐seq data with a public database, which was further verified by a series of in vitro experiments and immunohistochemistry.ConclusionWe demonstrated the single‐cell characteristics of AFPGC and that DKK1 facilitates AFP expression and malignancy.