Background: Hepatocellular carcinoma (HCC) is one of the most highly malignant tumors. Liver tumor-initiating cells (LTICs) have been considered to contribute to HCC progression and metastasis. ATP-citrate lyase (ACLY), as a key enzyme for de novo lipogenesis, has been reported to be upregulated in various tumors. However, its expression and role in HCC and LTICs remain unknown. Methods: The expressions of ACLY in HCC tissues were detected by quantitative real-time PCR (qRT-PCR), Western blotting and immunohistochemistry. Kaplan-Meier curves and Chi-square test were used to determine the clinical significance of ACLY expression in HCC patients. A series of assays were performed to determine the function of ACLY on stemness, migration and invasion of HCC cells. Luciferase reporter assay, Western blotting and immunoprecipitation were used to study the regulation of the Wnt/β-catenin signaling by ACLY. Rescue experiments were performed to investigate whether β-catenin was the mediator of ACLY-regulated stemness and migration in HCC cells. Results: ACLY was highly expressed in HCC tissues and LTICs. Overexpression of ACLY was significantly correlated with poor prognosis, progression and metastasis of HCC patients. Knockdown of ACLY remarkably suppressed stemness properties, migration and invasion in HCC cells. Mechanistically, ACLY could regulate the canonical Wnt pathway by affecting the stability of β-catenin, and Lys49 acetylation of β-catenin might mediate ACLY-regulated β-catenin level in HCC cells. Conclusions: ACLY is a potent regulator of Wnt/β-catenin signaling in modulating LTICs stemness and metastasis in HCC. ACLY may serve as a new target for the diagnosis and treatment of HCC.