Background <p>Lactate, traditionally regarded as a metabolic waste product of glycolysis, has recently emerged as a critical signaling molecule and epigenetic modifier via protein lactylation. However, the functional consequences of histone lactylation in cancer progression remain poorly understood.</p> Methods <p>We integrated transcriptomic data from TCGA and GEO (GSE188900) with bulk and single-cell RNA-seq analyses to identify lactylation-associated histone variants involved in esophageal squamous cell carcinoma (ESCC). Functional assays, immunoprecipitation (IP), western blotting (WB), chromatin immunoprecipitation (ChIP)-qPCR, and luciferase reporter assays were employed to investigate the role of H2BC9 and its lactylation in ESCC. A xenograft tumor model was used to validate in vivo relevance.</p> Results <p>We identified H2BC9, a histone H2B variant, as a lactylation-associated oncogene that is overexpressed in ESCC and correlates with poor prognosis. Lactate stimulation induced H2BC9 lactylation, particularly at lysine 44 (K44), as confirmed by mass spectrometry and IP-WB assays. Mechanistically, K44 lactylation of H2BC9 enhanced the transcriptional activity of Wnt7b, leading to activation of the Wnt/β-catenin pathway. Mutation of K44 (K44R) abolished H2BC9 lactylation and significantly impaired its ability to promote ESCC cell proliferation and Wnt7b transcription, as demonstrated by ChIP-qPCR and dual-luciferase assays. In vivo, H2BC9 K44R-expressing cells exhibited reduced tumor growth in xenograft models. Furthermore, H2BC9 expression was associated with an immunosuppressive tumor microenvironment and chemoresistance.</p> Conclusion <p>Our study reveals a novel regulatory axis in which H2BC9 K44 lactylation activates Wnt7b transcription and downstream Wnt/β-catenin signaling, driving ESCC progression. Targeting H2BC9 lactylation may offer a promising therapeutic strategy to overcome tumor growth and immune evasion in ESCC.</p>