Aim: To explore fecal short-chain fatty acids and neurotransmitter alterations in a mouse–glioma model and glioma patients. Methods: Liquid chromatography–mass spectrometry and 16S rRNA-sequencing from fecal samples were performed to measure metabolite levels and taxa abundance in mice/humans. Mice underwent GL261 implantation with/without temozolomide. Glioma patients were compared with healthy controls. Results: Glioma altered several short-chain fatty acids and neurotransmitter levels. Reduced 5-hydroxyindoleaceic acid and norepinephrine levels were seen in mice and humans. Interestingly, temozolomide treatment abrogates the effects of glioma on fecal metabolites. Conclusion: Our findings demonstrate the interplay between glioma and the gut–brain axis. Further work is required to identify pathways within the gut–brain axis by which glioma influences and promotes the modulation of fecal metabolites and microbiome.