Giant cell tumor of the bone (GCTB) is an osteolytic neoplasm of uncertain biological behavior that affects the epiphyseal ends of long bones in young adults. This study describes for the first time the ¹H NMR untargeted metabolic profiling of tissues from GCTB, which is clearly different from tissue controls. Applying univariate and multivariate analysis, fourteen differential metabolites were determinate and the major disturbed metabolic pathways included glycero-phospholipid metabolism and amino acid biosynthesis. These metabolic differences contribute to understand the molecular basis of GCTB and represent potential markers with future clinical applications.