The aim of the present study was to investigate the role of D3 receptor on intraocular pressure regulation using WT and KO D3R−/− mice. Both mice were used with normal eye pressure or steroid-induced ocular hypertension. As measured by tonometry, the topical application of 7-OH-DPAT, a dopamine D3-preferring receptor agonist, significantly decreased, in a dose-dependent manner, the intraocular pressure in WT mice both in an ocular normotensive group and an ocular hypertensive group. Pretreatment with U-99194A, a D3 receptor antagonist, reverted 7-OH-DPAT induced ocular hypotension in WT mice. No change of intraocular pressure was observed after topical application of 7-OH-DPAT in KO D3R−/− mice. PCR analysis demonstrated the presence of all dopamine receptor genes in eye tissues obtained from WT mice, and the lack of D3R mRNAs in KO mice. The present study identified the D3R subtype as the most important receptor of the dopaminergic system to modulate intraocular pressure with relevant implications for glaucoma that represents one of the most crippling optic neuropathies.