To investigate the effects and mechanism of anlotinib intervention on glycolysis, proliferation, colony formation and migration of human lung cancer NCI-H226 cells, to provide reference for in vitro test of lung cancer. Human lung cancer NCI-H226 cells were divided into control group which is without intervention, experimental group involving 10, 20, 40 μmol/L anlotinib, and positive control group involving 20 μg/mL cisplatin. The cell counting kit-8 (CCK-8), 5-acetylene-2′ deoxyuracil riboside (EdU) staining, plate clone formation, lactic acid detection kit, glucose detection kit, Transwell assay, RT-qPCR and Western blotting (WB) assays were used to detect proliferation viability, proliferation, colony-formation ability, lactic acid content, glucose consumption level, cell migration number and expression level of related factor levels. The proliferation activity, proliferation rate, clone formation rate, lactate content, glucose consumption level, N-cadherin, vimentin and fibronectin (FN) of NCI-H226 cells in control group, experimental group and positive drug group. There were significant differences in mRNA and protein expression levels between groups (P<0.05). The cell proliferation activity of 20 and 40 μmol/L anlotinib and positive drug groups was higher than that of control group (P<0.05). The cell proliferation rate, clone formation rate, lactate content, glucose consumption level and mRNA and protein expression levels of N-cadherin, vimentin and FN in the experimental group and the positive drug group were higher than those in the control group (P<0.05). Anlotinib can significantly inhibit glycolysis, proliferation, colony formation and migration of human lung cancer NCI-H226 cells.