Background: Hepatocellular carcinoma (HCC) is the most common malignancy of liver cancer. Calcium ions/calmodulins stimulate protein kinase kinases β (CaMKKβ) is a multifunctional protein kinase that is overexpressed in many types of cancer. This study aims to investigate the effect of CaMKKβ interference on HCC in HepG2 cells and transplanted tumor mice.Methods: CaMKKβ gene was knocked out in HepG2 cells as an experimental group, empty vector lentivirus as a negative control (NC) group, and untreated HepG2 cells as a control group. Cell proliferation, cell cycle, apoptosis, invasion, and glycolysis potential assays were conducted, respectively. In addition, the expression of PI3K, p-PI3K, AKT, and p-AKT was quantified by Western blot. Finally, the effect of CaMKKβ in vivo was investigated using a xenograft model. Results: CaMKKβ knockdown significantly suppressed HepG2 cell proliferation, cell cycle, invasion, EMT, and glycolysis, promoted cell apoptosis, and reduced the expression of hexokinase 2 (HK2), pyruvate kinase M (PKM2), and lactate dehydrogenase A (LDHA), p-PI3K, and p-AKT. Post the addition of AKT highly expression plasmid, glucose uptake, lactic acid production, and cell proliferation decreased, accompanied by an increase in apoptosis, which were substantially reversed. Notably, xenograft model experiments in vivo also confirmed that CaMKKβ knockdown inhibited HCC growth.Conclusions: CaMKKβ knockdown inhibited cell proliferation, invasion, and glycolysis through the PI3K/AKT pathway, heightened apoptosis, thus promoting the development of HCC. This might be a potential target for the diagnosis and treatment of HCC.