Background <p>Transfer RNA-derived small RNAs (tsRNAs) have been recognized as important regulators in human cancers. However, the role of tumor-suppressive tsRNAs in esophageal squamous cell carcinoma (ESCC) has not been thoroughly characterized. This study sought to elucidate the function of tsRNA-Gln-i-0061 (itRF-GlnCTG) in ESCC and its underlying molecular mechanism.</p> Materials <p>Bioinformatic approaches were employed to characterize the expression profile of tsRNAs in ESCC. Quantitative real-time PCR (qRT-PCR) and in situ hybridization (ISH) was applied to validate its expression levels in clinical samples. Functional assays, including proliferation and migration experiments, were conducted to detect the function of itRF-GlnCTG in ESCC. To identify downstream targets, RNA immunoprecipitation (RIP), biotin pull-down and mass spectrometry were performed. Additionally, the interaction between itRF-GlnCTG, the m6A reader RBMX, and reticulocalbin-1 (RCN1) mRNA was examined to uncover the regulatory mechanism.</p> Results <p>We identified itRF-GlnCTG as a downregulated tsRNA in ESCC tissues, and its elevated expression level correlated with better patient outcomes. Functionally, itRF-GlnCTG inhibited the growth, motility, and adhesion capabilities of ESCC cells. Mechanistically, itRF-GlnCTG acted as a molecular decoy by competitively binding to the m6A reader protein RBMX. This interaction impaired RBMX's SUMOylation, sequestered it in the cytoplasm, and thereby disrupted RBMX-mediated, m6A-dependent stabilization of RCN1 mRNA, leading to suppressed RCN1 expression. The combination of serum itRF-GlnCTG with CEA and CA199 showed high diagnostic efficacy for ESCC (AUC = 0.94).</p> Conclusions <p>Our results uncover a novel tumor-suppressive function of itRF-GlnCTG in ESCC by targeting the RBMX-m6A axis to destabilize oncogenic RCN1 mRNA. This study underscores the diagnostic utility of itRF-GlnCTG and reveals a new tsRNA-m6A regulatory pathway in ESCC.</p>