AbstractAnlotinib, as a promising oral small-molecule antitumor drug, its role in glioma has been only reported in a small number of case reports. Therefore, anlotinib has been considered as a promising candidate in glioma. The aim of this study was to investigate the metabolic network of C6 cells after exposure to anlotinib and to identify anti-glioma mechanism from the perspective of metabolic reprogramming. Firstly, CCK8 method was used to evaluate the effects of anlotinib on cell proliferation and apoptosis. Secondly, ultra-high performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS)-based metabolomic and lipidomic were developed to characterize the metabolite and lipid changes in cell and cell culture medium (CCM) caused by anlotinib in the treatment of glioma. As a result, anlotinib had concentration-dependent inhibitory effect with the concentration range. In total, twenty-four and twenty-three disturbed metabolites in cell and CCM responsible for the intervention effect of anlotinib were screened and annotated using UHPLC-HRMS. Altogether, seventeen differential lipids in cell were identified between anlotinib exposure and untreated groups. Metabolic pathways, including amino acid metabolism, energy metabolism, ceramide metabolism, and glycerophospholipid metabolism, were modulated by anlotinib in glioma cell. Overall, anlotinib has an effective treatment against the development and progression of glioma, and these remarkable pathways can generate the key molecular events in cells treated with anlotinib. Future research into the mechanisms underlying the metabolic changes is expected to provide new strategies for treating glioma.