Background <p>Super-enhancer-associated long non-coding RNAs (lncRNAs) have been shown to play key roles in the occurrence and development of malignant tumors, including esophageal squamous cell carcinoma (ESCC), yet their precise molecular mechanisms remain elusive.</p> Methods <p>ChIP-Seq, dual-luciferase reporter assays, HiChIP-Seq, and ChIP-qPCR were performed to investigate the transcriptional regulation mechanism of MIR205HG. The functions and downstream signal transduction mechanisms of MIR205HG in ESCC were explored by a series of in vitro and in vivo assays. Furthermore, comprehensive bioinformatics methods were used to analyze its correlation with ESCC patient survival.</p> Results <p>Here, we identified and characterized MIR205HG, a lncRNA driven by super-enhancer, as a crucial oncogene in ESCC. MIR205HG was up-regulated in SCCs and its high expression correlated with poor clinical outcomes. Both TP63 and KLF5, two important master transcription factors in ESCC, could simultaneously occupy the super-enhancer region at the <i>MIR205HG</i> locus to promote its transcription and overexpression. MIR205HG is essential for ESCC proliferation, migration, invasion, and the growth of xenograft tumors in vitro and in vivo. Mechanistically, MIR205HG directly bound PTBP3 and acted as a molecular scaffold to promote HIF-1α translation, leading to enhanced cellular glycolysis via up-regulating the expression of HK2 and LDHA. Moreover, survival and pseudotime analyses of ESCC scRNA-seq data revealed a significant positive correlation between MIR205HG/PTBP3 signaling and the stemness and malignancy of ESCC cells. Finally, we showed that specifically targeting MIR205HG-SE using a CRISPR interference method resulted in potent suppressive effects on ESCC malignant phenotypes.</p> Conclusion <p>We identified a pivotal oncogenic super-enhancer-driven lncRNA, MIR205HG, which interacts with PTBP3 to promote glycolysis in ESCC. It may serve as a promising prognostic biomarker and therapeutic target for patients.</p>