AIM: To explore the effect of the Notch signaling pathway on retinal ganglion cells (RGCs) and optic nerve in rats with acute ocular hypertension (OH). METHODS: Totally 48 Sprague-Dawley (SD) rats were included, among which 36 rats were selected to establish acute OH models. OH rats received a single intravitreal injection of 2 μL phosphate buffered solution (PBS) and another group of OH rats received a single intravitreal injection of 10 μmol/L γ-secretase inhibitor (DAPT). Quantitative real-time polymerase chain reaction (qPCR) and Western blot assay were adopted to determine the mRNA level of Notch and the protein levels of Notch, Bcl-2, Bax, caspase-3, and growth-associated protein 43 (GAP-43). The RGC apoptosis conditions were assessed by TUNEL staining. RESULTS: The OH rats and PBS-injected rats had increased expression levels of Notch1, Bax, caspase-3, and GAP-43, decreased expression levels of Bcl-2, and increased RGC apoptosis, with severer macular edema and RGCs more loosely aligned, when compared with the normal rats. The DAPT-treated rats displayed increased expression levels of Notch1, Bax, caspase-3, and GAP-43, decreased expression levels of Bcl-2, and increased RGC apoptosis, in comparison with the OH rats and PBS-injected rats. RGCs were hardly observed and macular edema became severe in the DAPT-treated rat. CONCLUSION: The Notch signaling pathway may suppress the apoptosis of retinal ganglion cells and enhances the regeneration of the damaged optic nerves in rats with acute OH.