The mechanisms underlying metastasis of colorectal cancer (CRC) remain unclear. C14orf159 is a mitochondrial matrix protein converting D-glutamate to 5-oxo-D-proline. Other metabolic functions of C14orf159, especially on mitochondrial metabolism, and its contribution to CRC metastasis, are not elucidated. Metabolome analysis by gas chromatography-mass spectrometry, RNA-sequencing analysis, flow cytometry, migration and invasion assay, sphere-formation assay using C14orf159-knockout and -stable expressing cells, immunohistochemistry of C14orf159 in human CRC specimens, and xenograft experiments using Balb/c nude mice were conducted. C14orf159 maintained the mitochondrial membrane potential of human CRC cells, and its involvement in amino acid and glutathione metabolism was demonstrated. In human CRC specimens, a decrease in C14orf159 expression at the invasive front of the tumour and in metastasis was determined. C14orf159 was also shown to attenuate the migration, invasion, and spheroid growth of CRC cells in vitro and colorectal tumour growth and metastasis in vivo. Mechanistically, C14orf159 reduced the expression of genes involved in CRC metastasis, including members of the Wnt and MMP family, by maintaining the mitochondrial membrane potential. Our findings link mitochondrial membrane potential to Wnt/β-catenin signalling and reveal a previously unrecognised function of the mitochondrial matrix protein C14orf159 as a suppressor of CRC metastasis.