Centromere protein L (CENPL) is involved in the mitotic process of eukaryotic cells and the development of various types of cancer. However, its role in hepatocellular carcinoma (HCC) remains unclear. This study aimed to investigate the expression and clinical significance of CENPL in HCC, and explore its involvement in regulating HCC cell proliferation, apoptosis, cell cycle, and glycolysis both in vivo and in vitro. CENPL expression was analyzed in HCC and normal liver tissues using The Cancer Genome Atlas, Gene Expression Omnibus mining, real-time quantitative polymerase chain reaction, and immunohistochemistry. Functional assays were used to assess the role of CENPL in HCC cell proliferation, apoptosis, cell cycle, and glycolysis. The potential pathways underlying the regulatory effects of CENPL, as well as the expression of mitogen-activated protein kinase (MAPK) signaling pathway-related molecules and markers of proliferation and glycolysis were investigated. CENPL was significantly upregulated in HCC tissue and associated with multiple clinicopathological features and poor patient prognosis. Univariate and multivariate analyses demonstrated that CENPL may serve as an independent prognostic factor for HCC. Upregulation of CENPL in HCC regulated tumor proliferation and glycolytic processes. Mechanistic studies revealed that differentially expressed genes between the CENPL-overexpressing and control groups were mainly concentrated in the MAPK signaling pathway. Pathway inhibition analysis indicated that CENPL activated the MEK1/2-ERK1/2 signaling pathway to promote proliferation and glycolysis in HCC cells. This study elucidated the role of CENPL in regulating cell proliferation, apoptosis, cell cycle, and glycolysis in HCC. CENPL may represent a therapeutic target and prognostic biomarker for HCC.Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.