Gonadotropin-releasing hormone (GnRH) is a ten-amino acid peptide hormone that plays pivotal roles in reproduction in vertebrates and octopus. Recently, six GnRH forms (t-GnRH-3-8) and four GnRH receptor subtypes (Ci-GnRHR-1-4) were identified in the protochordate, Ciona intestinalis. In this study, we show the functional modulation of Ci-GnRHR-1 via heterodimerization with the orphan receptor subtype, Ci-GnRHR-4. The dimerization between Ci-GnRHR-1 and R-4 was detected by co-immunoprecipitation and immunoblot analysis. Binding assays confirmed the binding of t-GnRHs to Ci-GnRHR-1 but not to R-4, and verified no alternation in ligand-binding affinity between Ci-GnRHR-1 homodimer and Ci-GnRHR1&4 heterodimer. The heterodimer was found to stimulate the elevation of intracellular calcium, time-extension of ERK phosphorylation, and Up-regulation of cell proliferation, all in a ligand specific manner, compared with the Ci-GnRHR-1 homodimer. In combination, these results indicated that Ci-GnRHR-4 is not an inactive receptor, but a modulatory factor for Ci-GnRHR-1 in C. intestinalis.