A cerebral upregulation of the translocator protein (TSPO), a biomarker of glial activation, has been reported in fibromyalgia subjects (FMS). The TSPO binding affinity is genetically regulated by the Ala147Thr polymorphism in the TSPO gene (rs6971) and allows for a subject classification into high and mixed/low affinity binders, HABs and MLABs. Aim of the present multimodal neuroimaging study was to examine the associations of the TSPO polymorphism with: I) conditioned pain modulation, II) expectancy-modulated pain processing assessed during functional magnetic resonance imaging, and III) the concentration and balance of glutamate and γ-aminobutyric acid (GABA) in the rostral anterior cingulate cortex (rACC) and thalamus using proton magnetic resonance spectroscopy in FMS (n = 83) and healthy controls (HC, n = 43). The influence of TSPO on endogenous pain modulation presented in the form of TSPO HABs, as opposed to MLABs, displaying less efficient descending pain inhibition and expectancy-induced reduction of pain. TSPO HABs in both groups (FM and HC) were found to have higher thalamic glutamate concentrations and exhibit a pattern of positive correlations between glutamate and GABA in the rACC, not seen in MLABs. Altogether, our findings point to TSPO-related mechanisms being HAB-dependent, brain region-specific, and non-FM-specific, although in FMS the disadvantage of an aberrant pain regulation combined with a HAB genetic set-up might hamper pain modulation more strongly. Our results provide evidence for an important role of TSPO in pain regulation and brain metabolism, thereby supporting the ongoing drug development targeting TSPO-associated mechanisms for pain relief.