Cancer stem cells (CSCs) play an essential role in tumor initiation and therapy resistance. Histone lactylation as a novel epigenetic modification could regulate the gene transcription process during tumor progression. Nevertheless, researches have not well examined its role in maintaining CSC properties. Our study identified Minichromosome maintenance complex component 7 (MCM7) as a candidate gene in Hepatocellular carcinoma (HCC) with diagnostic and prognostic values, and Real-time quantitative PCR (qRT-PCR), Western blot (WB), and Immunohistochemistry (IHC) assays ascertained its obviously higher expressions in HCC cells and tissues. Ectopic of MCM7 could increase the expression of CSC-related genes and enhance spheroid both in size and in number. Suppression of MCM7 could strengthen the efficacy of radiotherapy verified by Cell counting kit-8 (CCK-8) and colony formation assays. The subcutaneous xenograft model indicated that suppression of MCM7 could inhibit CSC properties and the efficacy of radiotherapy in vivo. Mechanistically, histone lactylation could facilitate MCM7 expression, and both messenger RNA (mRNA) and protein level of MCM7 expression presented an obvious decrease due to 2-DG (glycolysis inhibitor) treatment and an obvious increase due to Rotenone (glycolysis activator) treatment. Rescue experiments verified that histone lactylation was necessary for MCM7 to promote CSC properties and radio-resistance in HCC. Arsenic trioxide (ATO) targeting MCM7 could inhibit the CSC phenotypes and enhance the efficacy of radiotherapy in vivo and in vitro. Collectively, histone lactylation could transcriptionally activate MCM7 to accelerate proliferation and radio-resistance through enhancing CSC properties. ATO targeting MCM7 could inhibit CSCs phenotypes and synergistically increase the efficacy of radiation therapy.Copyright © 2025 Elsevier Inc. All rights reserved.