<p>Glycine C-acetyltransferase (GCSH), a component of the mitochondrial glycine cleavage system, has been previously linked to cuproptosis or tumor progression, but its role in colorectal cancer (CRC) remains incompletely understood. We analyzed GCSH expression across multiple CRC datasets, including the TCGA dataset (<i>n</i> = 689) and four independent GEO datasets (total <i>n</i> = 709), and validated findings in clinical tissues, blood samples, and CRC cell lines. Functional roles of GCSH were assessed using CCK-8, wound healing, and Transwell assays. Cuproptosis was induce, and evaluated by flow cytometry, Western blotting, and measurement of intracellular Cu²⁺, ROS, and FDX1 levels. The regulatory relationship between GCSH and FDX1, as well as the involvement of the PI3K/AKT pathway, was explored through rescue experiments and molecular docking analysis. The analysis of Bulk RNA-seq data and scRNA-seq datasets revealed the expression and the biological function of GCSH in CRC. The validation experiments confirmed that GCSH was consistently overexpressed in CRC tissues and blood samples, and its expression correlated with distant metastasis. GCSH knockdown inhibited cell viability, migration, and invasion, with minimal effects on apoptosis. Mechanistically, GCSH suppressed cuproptosis by downregulating FDX1 protein and reducing intracellular Cu²⁺ and ROS accumulation. Molecular docking suggested a potential interaction between GCSH and FDX1. Furthermore, GCSH was identified as a downstream effector of PI3K/AKT pathway, mediating its protective effect against cuproptosis. GCSH promotes CRC progression and confers resistance to cuproptosis via the PI3K/AKT-GCSH/FDX1 axis. These findings identify GCSH as a novel regulator of cuproptosis and a potential therapeutic target in CRC.</p>