Glioma is the most prevalent malignant brain tumor. A comprehensive analysis of the glioma metabolome is still lacking. This study aims to explore new special metabolites in glioma tissues. A non-targeted human glioma metabolomics was performed by UPLC-Q-TOF/MS. The gene expressions of 18 enzymes associated with 3'-phosphoadenylate (pAp) metabolism was examined by qRT-PCR. Those enzymes cover the primary metabolic pathway of pAp. We identified 15 new metabolites (13 lipids and 2 nucleotides) that were significantly different between the glioma and control tissues. Glycerophosphatidylcholine [PC(36:1)] content was high and pAp content was significantly low in the control brain (p < 0.01). In glioma tissues, PC(36:1) was not detected and pAp content was significantly increased. The gene expressions of 3'-nucleotidases (Inositol monophosphatase (IMPAD-1) and 3'(2'),5'-bisphosphate nucleotidase 1(BPNT-1)) were dramatically down-regulated. Meanwhile, the gene expression of 8 sulfotransferases (SULT), 2 phosphoadenosine phosphosulfate synthases (PAPSS-1 and PAPSS-2) and L-aminoadipate-semialdehyde dehydrogenase-phosphopante-theinyl transferase (AASDHPPT) were up-regulated. PC(36:1) absence and pAp accumulation are the most noticeable metabolic aberration in glioma. The dramatic down-regulation of IMPAD-1 and BPNT-1 are the primary cause for pAp dramatic accumulation. Our findings suggest that differential metabolites discovered in glioma could be used as potentially novel therapeutic targets or diagnostic biomarkers and that abnormal metabolism of lipids and nucleotides play roles in the pathogenesis of glioma.