Gastric cancer (GC) is the third most lethal and fifth most common cancer in the world. In a variety of cancers, the hexokinase domain component 1 (HKDC1) is carcinogenic. This study was to investigate into how HKDC1 contributes to the development and progression of GC. Three different datasets (GSE103236, GSE13861, and GSE55696) were extracted from the Gene Expression Omnibus (GEO) database and then analyzed using the sva package. The R software was used to identify 411 differentially expressed genes (DEGs) in the pooled dataset. We discovered 326 glycolysis-related genes (glyGenes) in the cancer genome atlas-stomach adenocarcinoma (TCGA-STAD) cohort using gene set enrichment analysis set (GSEA). HKDC1 is one of the most prevalent glyGenes in GC tumor tissues and cells, as seen in the Venn diagram. According to the results of the Cell Count Kit-8 assay, the proliferation of AGS and MKN-45 cells decreased when HKDC1 was knocked down. Lack of HKDC1 in cells enhanced oxygen consumption and decreased glycolytic protein expression while suppressing glucose absorption, lactate production, ATP level, and extracellular acidification ratio. As an oncogene in gastric cancer development, HKDC1 influences cell proliferation and glycolysis.Copyright © 2023 Chen Yu et al.