tRNA-derived fragments (tRFs) are a class of regulatory non-coding RNAs that play essential biological functions in cancer and stress-induced diseases. Several lines of evidence suggest that 5′-tRF-GlyGCC participates in tumor progression; however, its molecular mechanisms remain unclear. In this study, we explored the function of 5′-tRF-GlyGCC in breast cancer (BC) progression and studied the related potential molecular mechanisms. 5′-tRF-GlyGCC expression increased in human BC, and it promoted the proliferation, migration, and invasion of BC cells in vitro and tumor growth and metastasis in vivo. 5′-tRF-GlyGCC was found for the first time to bind directly to fat mass and obesity-associated proteins, and increase the activity of FTO demethylase, reducing eIF4G1 methylation, inhibiting autophagy, and promoting BC proliferation and metastasis. These findings suggest that 5′-tRF-GlyGCC might be a therapeutic target for treating BC. • 5′-tRF-GlyGCC promotes breast cancer metastasis. • 5′-tRF-GlyGCC directly combines with FTO to increase its demethylase activity. • 5′-tRF-GlyGCC reduces methylation of eIF4G1 and inhibits autophagy. • 5′-tRF-GlyGCC might be a therapeutic target for breast cancer. [ABSTRACT FROM AUTHOR]