Objectives: Enhanced stress responsiveness is an important pathophysiological factor in irritable bowel syndrome (IBS), suggesting the presence of a dysregulated hypothalamic-pituitary-adrenal (HPA) axis. A possible mechanism involves maladaption of the feedback mechanism of the HPA axis. We hypothesized that hippocampus, a key brain region providing inhibitory feedback to the HPA axis, would exhibit reduced excitatory glutamatergic neurotransmission and reduced N-acetyl-aspartate (NAA; a marker of neuronal integrity) levels in IBS patients. Methods: In this preliminary study, proton magnetic resonance spectroscopy was used to quantify absolute concentrations of metabolites in bilateral hippocampi of 15 IBS patients without significant psychiatric comorbidity and 15 age-matched controls. Results: The main finding was a reduction in hippocampal glutamate-glutamine (Glx) in IBS patients. Furthermore, Glx concentrations were inversely related to emotional stress indicators in patients only. No difference was found between subject groups for other metabolite concentrations, including NAA. However, an elevated myo-inositol (mI)/NAA ratio was found in IBS patients. Conclusions: Our results provide preliminary evidence for the presence of abnormal hypofunction of hippocampal glutamatergic neurotransmission in IBS patients without psychiatric comorbidity, possibly as a result of the chronic pain. This supports the notion of an imbalance in regulatory brain regions in this subgroup of IBS patients. The inverse relationship between Glx and emotional stress indicators is in agreement with the inhibitory role of hippocampus on the stress system and suggests a sensitization of the mechanism to emotional arousal. The elevated mI/NAA ratio in IBS patients further suggests the presence of hippocampal glial proliferation and remodeling.