The aim of this study was to explore whether polymorphisms in TFAM gene are associated with Korean GC patient and discover the novel role of TFAM in GC. The genotypes of 2147 Korean participants (including 548 IGC and 393 DGC GC patients) were examined for 9 tag single-nucleotide polymorphisms (SNPs). Susceptibility to diffuse-type GC was strongly and significantly associated with several SNPs including rs10826175 (A>G) in the intergenic region (GG+GA versus AA, odds ratio = 1.396 P = 0.019) and rs1937 (G>C) in exon 1 (CC+GC versus GG, odds ratio = 1.287, P = 0.044). To validate the GC association with TFAM and how TFAM functions in GC, we silenced TFAM using siRNA and shRNA in GC cell lines. Downregulation of TFAM by siRNA changed cell morphology, decreased cell proliferation, and inhibit cell migration and invasion in MKN45 (diffuse type GC cell line). In order to gain insights of how these phenotype is influenced by TFAM downstream genes, RNA sequencing (RNA-seq) was performed. Differentially expressed genes (DEG) analysis was performed with scrambled siRNA treated control samples and TFAM knockdown samples. Finally, 101 genes were selected. Among 101 DEGs, 68 genes, which have gene information, Quantitative Real time PCR (qPCR) was performed to validate the expression level. Finally, top 10 genes were selected with following criteria: qPCR p-value> 0.05 at 6 h dataset, qPCR p-value <0.05 and FC> 3 at 24 h dataset. Among top 10 DEGs, when CCDC108 and PCK1 expression were rescued in TFAM knockdown phenotype in MKN45. When both genes were rescued respectively, it alleviates the phenotypic effects of TFAM knockdown in cell proliferation and morphological change of MKN45. Among TFAM downstream genes that may be responsible for functional role in promoting cancer cell growth and invasion, TFAM-CCDC108-PCK1 may play pivotal roles in GC.