Background <p>Vasoactive Intestinal Peptide (VIP) is a pleiotropic neuropeptide regulating diverse cellular and physiological processes. Its functions are primarily mediated through two G protein–coupled receptors, VPAC1 and VPAC2. The aim of this study was to perform an integrative analysis of VPAC receptor signalling, encompassing receptor–G protein coupling, second messenger production, kinase activation and transcriptional responses in T cells.</p> Experimental approach <p>Receptor interactions with Gα subunits were analysed using BRET assays. Stable Jurkat T-cell lines overexpressing VPAC1 (J-OEV1) or VPAC2 (J-OEV2) were generated. VPAC-dependent intracellular signalling in these cells was assessed by measuring cAMP and Ca²⁺ levels, performing phospho-kinase arrays and Western blot analyses, and evaluating immune mediator expression, cell viability, and proliferation.</p> Results <p>VPAC1 showed interaction with both Gαs and Gαq subunits, whereas VPAC2 preferentially interacted with Gαs. In Jurkat cells, both receptors overexpression enhanced cAMP signalling, while increased Ca²⁺ responses were restricted to VPAC1. In both J-OEV1 and J-OEV2 cells, VIP treatment reduced phosphorylation of inflammatory kinase-associated proteins. Overexpression of either receptor induced distinct basal transcriptional profiles of transcription factors and cytokines, which were further modulated by CD3/CD28-activation and VIP. While proliferation was not altered with overexpression, J-OEV2 showed a reduced redox metabolism at 72&#xa0;h.</p> Conclusion <p>This study aimed to identify functional differences between VPAC1 and VPAC2 signalling and reveals reproducible subtype-specific differences at early signalling. In Jurkat cells, both receptors induce a shift in its basal state; however, changes do not persist during TCR-driven activation, resulting in largely convergent effector responses.</p>