Elevated intraocular pressure (IOP) is the main recognized risk factor of glaucoma. To investigate the contribution of dopaminergic and serotonergic systems in IOP regulation, we used cabergoline, a mixed dopamine and serotonin agonist, in C57BL/6J WT and dopamine D3 receptor knock-out (D3R(-/-)) mice with normal eye pressure or steroid-induced ocular hypertension. Furthermore, we studied the structural basis of the cabergoline-mediated activation of the dopaminergic and serotonergic systems by molecular modeling. Topical application of cabergoline, significantly decreased, in a dose-dependent manner, the intraocular pressure in WT mice, both in an ocular normotensive group (-9, -5 and -2mmHg with 5%, 1%, and 0.1%, respectively) and an ocular hypertensive group, with a prolonged effect in this latter group. No change of intraocular pressure was observed after topical application of cabergoline in D3R(-/-) mice. We modeled and optimized, with molecular dynamics, structures of hD3, h5HT1A and h5HT2A-C receptors; thereafter we carried out molecular docking of cabergoline. Docking revealed that binding of cabergoline into D3 and 5HT1A receptors is associated with a better desolvation energy in comparison to 5HT2A-C binding. In conclusion, the present study support the hypothesis that dopaminergic system is pivotal to regulate IOP and that D3R represents an intriguing target in the treatment of glaucoma. Furthermore, the structure-based computational approach adopted in this study is able to build and refine structure models of homologous dopaminergic and serotonergic receptors that may be of interest for structure-based drug discovery of ligands, with dopaminergic selectivity or with multi-pharmacological profile, potentially useful to treat optic neuropathies.