AbstractChronic pain is a world-wide clinical challenge. Response to analgesic treatment is limited and difficult to predict. Functional MRI has been suggested as a potential solution. However, while most analgesics target specific neurotransmission pathways, functional MRI-based biomarkers are not specific for any neurotransmitter system, limiting our understanding of how they might contribute to predict treatment response. Here, we sought to bridge this gap by applying Receptor-Enriched Analysis of Functional Connectivity by Targets to investigate whether neurotransmission-enriched functional connectivity mapping can provide insights into the brain mechanisms underlying chronic pain and inter-individual differences in analgesic response after a placebo or duloxetine. We performed secondary analyses of two openly available resting-state functional MRI data sets of 56 patients with chronic knee osteoarthritis pain who underwent pre-treatment brain scans in two clinical trials. Study 1 (n = 17) was a 2-week single-blinded placebo pill trial. Study 2 (n = 39) was a 3-month double-blinded randomized trial comparing placebo to duloxetine, a dual serotonin–noradrenaline reuptake inhibitor. Across two independent studies, we found that patients with chronic pain present alterations in the functional circuit related to the serotonin transporter, when compared with age-matched healthy controls. Placebo responders in Study 1 presented with higher pre-treatment functional connectivity enriched by the dopamine transporter compared to non-responders. Duloxetine responders presented with higher pre-treatment functional connectivity enriched by the serotonin and noradrenaline transporters when compared with non-responders. Neurotransmission-enriched functional connectivity mapping might hold promise as a new mechanistic-informed biomarker for functional brain alterations and prediction of response to pharmacological analgesia in chronic pain.