BACKGROUND: Enhanced glycolysis contributes to the chemotherapy resistance of colorectal cancer (CRC). However, whether tRNA-derived small RNAs (tsRNAs) regulate CRC oxaliplatin sensitivity through glycolysis-mediated histone lactylation remains unclear. METHODS: By analyzing RNA-seq data from CRC samples in the TCGA database, we identified a glucose metabolism-related tsRNA. Overexpression of tsRNA-08614 was investigated to explore its impact on CRC sensitivity to oxaliplatin. The targeting gene of tsRNA-08614 was validated through a dual-luciferase reporter assay. The specific molecular mechanism of tsRNA-08614 regulating CRC oxaliplatin sensitivity was further revealed by ChIP-seq and RNA-seq. RESULTS: We found a down-regulated tsRNA-08614 targeted glycolysis-related gene, which was associated with chemotherapy resistance. Overexpression of tsRNA-08614 promoted oxaliplatin sensitivity of CRC cells. tsRNA-08614 inhibited the expression of the target gene ALDH1A3 and reduced glycolysis, whereas the glycolytic inducer reversed the enhanced sensitivity caused by tsRNA-08614. Interference of tsRNA-08614 increased H3K18la and pan-Kla levels, while the lactate inhibitor partially suppressed these effects. Furthermore, overexpression of tsRNA-08614 reprogrammed the transcription of genes mediated by histone lactylation modification, with EFHD2 showing the most significant differential expression. EFHD2 inhibited the sensitivity of CRC cells to oxaliplatin by upregulating CMPK2 and enhancing mitochondrial function. Finally, we demonstrated that tsRNA-08614 enhanced sensitivity to oxaliplatin in CRC by reducing histone lactylation levels in vivo. CONCLUSION: tsRNA-08614 regulates ALDH1A3 to inhibit glycolysis and histone lactylation modification, thereby suppressing the transcriptional activity of EFHD2 and ultimately promoting the sensitivity of CRC to oxaliplatin. These findings suggest that tsRNA-08614 may represent a novel molecular target to combat oxaliplatin resistance in CRC chemotherapy.