This study aimed to investigate the associations of liquid-liquid phase separation (LLPS) and tumor stemness in hepatocellular carcinomas (HCC). LLPS-related genes were extracted from DrLLPS, LLPSDB and PhaSepDB databases. Stemness index (mRNAsi) was calculated based on the data from TCGA and Progenitor Cell Biology Consortium. Through some series of bioinformatics methods, we first found that stemness index mRNAsi was associated with worse survival outcomes, immune infiltration and therapy sensitivity in HCC. G2M checkpoint and DNA repair pathways were significantly activated with high mRNAsi. Totally, 71 differentially expressed LLPS genes in HCC were correlated with mRNAsi, and a mRNAsi-associated LLPS gene signature (KPNA2, EEF1E1 and ATIC) was identified to predict prognosis for HCC patients. mRNAsi-associated LLPS genes contributed to cluster HCC patients into four molecular clusters that markedly differed on survival, immune infiltration and therapy sensitivity. Further in vivo and in vitro experiments showed that EEF1E1 was highly expressed in HepG2 and HCCLM3 cells, and EEF1E1 silencing observably inhibited tumor cell growth, liver cancer stem cells (CSCs) markers (CD133, EpCAM and SOX2) expression, enhanced DNA damage marker γH2AX expression by activating PTEN/AKT pathway. EEF1E1 could undergo LLPS condensates, and roles of EEF1E1 on tumor cells were partly reversed after inhibiting LLPS using 1, 6-hexanediol. In conclusion, EEF1E1 was identified as a phase separation protein and involves in tumor stemness and DNA damage repair in HCC. EEF1E1 and its LLPS condensate may be novel targets to elaborate the underlying mechanisms of CSCs propagation in HCC.