5-Fluorouracil (5-FU) is a first-line chemotherapeutic agent for advanced gastric cancer (GC). However, its clinical efficacy is often undermined by the development of chemoresistance. Aberrant activation of oncogenic pathways, including autophagy, has been implicated in 5-FU resistance. Epigenetic modifications, such as 5-methylcytosine (m5C), are also recognized to modulate autophagy and contribute to chemoresistance, though the underlying molecular mechanisms remain poorly understood. In this study, we discovered that YBX1, an m5C reader protein, was significantly upregulated in 5-FU-resistant GC cell lines and patient tissues. Both in vitro and in vivo experiments demonstrated that YBX1 promoted autophagy in GC cells, thereby enhancing 5-FU resistance. Mechanistically, the transcription factor MAZ was found to bind to the YBX1 promoter, driving its transcriptional upregulation. YBX1, in turn, stabilized ATG9A mRNA via NSUN2-mediated m5C modification, thereby enhancing autophagic activity and conferring chemoresistance. Clinically, elevated YBX1 expression correlated with poor prognosis in patients with advanced GC undergoing 5-FU-based chemotherapy. These findings establish YBX1 as a key regulator of autophagy and 5-FU resistance in GC and highlight its potential as a novel therapeutic target for overcoming 5-FU resistance.