Glutaminase controls the first step in glutaminolysis, impacting bioenergetics, biosynthesis and oxidative stress balance. Two isoenzymes exist in humans, GLS and GLS2. GLS is considered prooncogenic and overexpressed in many tumours, while GLS2 may act as both prooncogenic or as a tumour suppressor. Glioblastoma cells usually lack GLS2 while express high GLS. We aimed to investigate how GLS2 expression modifies the metabolism of glioblastoma cells, looking for changes that may explain GLS2’s potential tumour suppressive role. We developed LN-229 human glioblastoma cells stably expressing GLS2 and performed isotope tracing using U-13C-glutamine and metabolomic quantification to analyse metabolic changes. Treatment with the GLS inhibitor CB-839 was also included to concomitantly inhibit endogenous GLS activity. GLS2 overexpression resulted in extensive metabolic changes, altering the TCA cycle in an isoenzyme-specific and previously unsuspected way, by upregulating part of the cycle but blocking the synthesis of the 6-carbon intermediates from acetyl-CoA. Expression of GLS2 caused downregulation of PDH activity through phosphorylation of S293 of PDHA1. GLS2 also altered nucleotide levels as well as induced the accumulation of methylated metabolites and methyl donor S-adenosyl methionine. These changes suggest that GLS2 may be a key regulator linking glutamine and glucose metabolism, also impacting nucleotides and epigenetics.