Complex interactions between serotonin (5-hydroxytriptamine) and orexin (hypocretin) systems in the CNS modulate diverse physiological processes, including mood regulation, energy homeostasis, and spontaneous physical activity. A portion of this interaction may be explained by G protein-coupled receptor oligomerization, which might influence agonist affinity, efficacy, trafficking, and signal transduction. Orexin receptor 1 (OX1) and 5-hydroxytriptamine 2C receptor (5-HT2C) co-express in diverse neuronal populations and are involved in pathological conditions such as anxiety and eating disorders, but their direct interaction has not been addressed. In this study, we aimed to elucidate the physical and functional interaction between OX1 and 5HT2C receptors in an in vitro heterologous expression system. We transiently transfected HEK-293T cells with cDNAs encoding the human OX1 and 5HT2C receptors, both modified to perform bioluminescence-based assays through the nanoluciferase binary technology (NanoBiT™). We assessed heteromer formation, both in presence and absence of orexin-A. Next, we evaluated whether OX1 presence modified 5HT2C coupling to mini-Gα proteins and/or β-arrestin. Finally, functional assays including measurement of agonist-induced calcium influx were conducted to assess downstream signalling upon receptor activation. Our results demonstrate the formation of heteromers between OX1 and 5-HT2C receptors, which is reduced upon orexin-A binding. This agonist-dependent alteration in heteromerization may modify the G-protein coupling profile and downstream signalling pathways. Understanding OX1-5HT2C receptor interactions provides novel insights into the physiological and pathological processes regulated by the orexin-serotonin axis.