<p>Intestinal immune activation and neuro-immune interactions have been proposed to contribute to the pathophysiology of functional dyspepsia (FD), but advanced characterization of the immune landscape is largely lacking. This study aimed to extensively characterize the duodenal and peripheral immune landscape in FD; to evaluate the potential of the duodenal microenvironment to activate spinal sensory neurons; and to assess immunomodulatory effects of high-dose proton pump inhibitor (PPI, 40 mg pantoprazole 2×/day) intervention in FD. Duodenal tryptase release (<i>P</i> = 0.0041) and CD45RA^- CD8⁺ T cells (<i>P</i> = 0.0079) were elevated in 30 prospectively recruited patients with Rome IV FD compared to 30 healthy controls (HC), and correlated with gastrointestinal (GI) symptoms. In vitro response rate of murine dorsal root ganglion neurons to duodenal biopsy supernatants of FD and HC was similar (<i>P</i> = 0.43), despite associations with GI symptoms but not duodenal mediator release. High-dose PPI improved GI symptoms (<i>P</i> = 0.0002) but increased duodenal mast cells (<i>P</i> = 0.044), systemic monocytes (<i>P</i> = 0.011) and α₄β₇⁺ gut-homing T cells (<i>P</i> = 0.0073) in FD. These limited immunological differences in patients with FD not taking PPI suggest an overestimation of the extent of immune activation in previous studies. Neuro-immune interactions can underly visceral pain sensation in FD, although presumably not mediated by mast cell tryptase. ClinicalTrials.gov registration: NCT04713969, 13/01/2021.</p>