Objective To investigate the effect and mechanism of axon guidance molecule semaphorin 3A (SEMA3A) on the apoptosis of retinal ganglion cells (RGCs) in diabetic rats. Methods Forty adult male SD rats were randomly divided into control group (normal feeding), model group (establishing diabetic retinopathy ［DR］ model by intraperitoneal injection of streptozotocin), si-SEMA3A group (intravitreal injection of 10 μL SEMA3A-siRNA lentivirus ［titer 1×10⁹ IU/mL］ after establishing DR model), and si-SEMA3A＋DAPT group (intravitreal injection of 10 μL SEMA3A-siRNA lentivirus ［titer 1×10⁹ IU/mL］ and 10 μL Notch1 pathway inhibitor N-(N-((3,5-difluorophenyl)acetyl)-L-alanyl-L-2-phenyl)glycine-1,1-dimethylethyl ester ［DAPT, 10 μmol/L］ after establishing DR model), with 10 rats in each group. Samples were collected after 12 weeks of treatment in each group; immunofluorescence staining by double labeling of SEMA3A and POU domain transcription factor 3a (Brn3a, a RGC marker) was used to detect the expression and localization of retinal SEMA3A protein; hematoxylin-eosin staining was used to detect the density of RGCs; terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) method was used to detect the apoptosis of RGCs; enzyme-linked immunosorbent assay (ELISA) was used to detect the expression levels of retinal inflammatory factors interleukin 6 (IL-6) and tumor necrosis factor α (TNF-α); and Western blotting was used to detect the expression levels of SEMA3A, Notch1, and cysteine aspartic acid specific protease 3 (caspase 3) in retinal tissues. Results The specific expression of SEMA3A in RGCs was confirmed by double-labeled immunofluorescence staining. Compared with the control group, the density of RGCs in the model group was significantly lower (P＜0.05). The density of RGCs in the si-SEMA3A group was significantly higher than that in the model group and si-SEMA3A＋DAPT group (both P＜0.05). No TUNEL-positive RGCs were detected in the retinal tissue of control rats. In the model group, there were more TUNEL-positive RGCs in the retinal tissue of rats, with a RGC apoptosis rate of (49.55±7.82) %. Compared with the model group, the apoptosis rate of the si-SEMA3A group was significantly lower (P＜0.05), while the apoptosis rate of the si-SEMA3A＋DAPT group was significantly higher than that of the si-SEMA3A group (P＜0.05). Compared with the control group, the expression levels of IL-6, TNF-α, SEMA3A, and caspase 3 in the model group were significantly higher, while the expression level of Notch1 was significantly lower (all P＜0.05). Compared with the model group, the expression levels of IL-6, TNF-α, SEMA3A, and caspase 3 were significantly lower in the si-SEMA3A group, while the expression level of Notch1 was significantly higher (all P＜0.05). The expression levels of IL-6, TNF-α, SEMA3A, and caspase 3 in the si-SEMA3A＋DAPT group were significantly higher than those in the si-SEMA3A group, while the expression level of Notch1 was significantly lower (all P＜0.05). Conclusion SEMA3A may be involved in the apoptosis of RGCs in diabetic rats via the Notch1 signaling pathway, and is expected to be a therapeutic target for DR.