BACKGROUND: Glutathione plays critical roles in detoxifying xenobiotics, cell signaling, cell death and the antioxidant defence in an emerging body of evidence, the most abundant intracellular low molecular weight thiol in tissues. However, all glutathione metabolism pertinent genes (GMPGs) expression and their diagnostic/prognostic/micropeptide potential analyses have not been investigated to perform in pan-cancers. METHODS: We gained GMPGs from the MsigDB 7.2, 12,123 samples were used to reveal the differentially expressed genes (DEGs) and the survival analysis in 32 types of cancers from TCGA, GTEx, and GEO datasets for the first time. All statistical analyses were performed by R for bioinformatics, such as DEGs, prognostic, diagnostic analysis, ceRNA, micropeptide prediction and immune infiltration. In addition, we utilized siRNA technology to target knockdown the expression of the G6PD gene in Huh7 hepatocellular carcinoma cells. RESULTS: G6PD was significantly expressed and poor prognosis in liver hepatocellular carcinoma (LIHC) and predicted RBM26-AS1 encoded micropeptide might target G6PD in LIHC. In vitro experiments show that G6PD knockout in Huh7 cells reduces their proliferation, migration, and invasion capabilities. CONCLUSIONS: We confirmed that G6PD played a crucial role in the occurrence and progression of LIHC. G6PD is positively associated with Th2 cells in LIHC, regulating immune responses in the immune system. We considered that micropeptide RBM26-AS1 might be a new player involved in LIHC by interacting with G6PD, might perform a key function in liver cancer.