We investigated whether P2X 7 antagonists rescue retinal ganglion cells (RGCs) in culture and after optic nerve crush (ONC) injury. Rats were sacrificed 7 days after retrograde labeling of RGCs with 4′,6-diamidino-2-phenylindole (DAPI), and the retinas were enzymatically dissociated in vitro and incubated with P2X 7 antagonists or agonists for 3 days. Adenosine triphosphate (ATP) and benzoylbenzoyl ATP were used as P2X 7 agonists, and oxidized ATP and brilliant blue G were used as P2X 7 antagonists. DAPI-positive and calcein-positive RGCs were counted to determine the number of living cells. We observed that RGCs were preserved when treated with P2X 7 antagonists, as compared with the controls. In contrast, P2X 7 agonists significantly decreased the number of viable RGCs. In vivo, P2X 7 antagonists at various doses were injected into the vitreous body immediately after ONC injuries in rats. Surviving RGCs were stained with anti-neuron-specific β-tubulin antibody in flat-mounted retinas. RGCs were observed to decrease to 61% of baseline 7 days after ONC injury, whereas RGCs were significantly preserved when P2X 7 antagonists were applied. When P2X 7 receptor expression was examined immunohistochemically in rat retinas after ONC, the retinal expression of the P2X 7 receptors was observed to be upregulated after ONC and peaked on day 3. Meanwhile, P2X 7 antagonists suppressed this upregulation. Collectively, these results suggest that P2X 7 antagonists prevent loss of RGCs after ONC, and that this protective effect is possibly mediated through suppressing the upregulation of retinal P2X 7 expression.