Objective To study the effect of down-regulation of Claudin-3 mediated by adeno-associated virus (AAV) of shRNA on the cultured retinal ganglion cells (RGCs) in vitro. Methods RGCs isolated from mouse eyes were divided into normal control group, AAV-shScramble group, and AAV-shClaudin-3 group. The RGCs in AAV-shScramble group and AAV-shClaudin3 group were treated with AAV-shScramble and AAV-shClaudin-3 respectively 24 hours after cell seeding. Dynamic live cell fluorescence microscopy was used to observe the transfection efficiency 96 hours after transfection. Immunofluorescent staining of β-tubulin was used to measure the length of RGCs′ axon. 4′, 6-diamidino-2-phenylindole staining was used to observe the nuclei of apoptotic cells. The mRNA level of Claudin-3 and VEGF was measured by real-time polymerase chain reaction. The protein levels of Claudin-3, vascular endothelial growth factor (VEGF), Bcl-2 and Caspase-3 was determined by Western blot. Results The positive transfection rate was more than 50% in both AAV-shScramble group and AAV-shClaudin-3 group. The length of RGCs' axon in AAV-shClaudin-3 group was shorter than that in normal control group and AAV-shScramble group (F=22 363.274,P＜0.05). Down-regulation of Claudin-3 accelerated RGCs' apoptosis with nuclei shrinkage, tapering, and nucleolus formation of apoptotic bodies. The mRNA levels of Claudin-3 and VEGF in AAV-shClaudin-3 group were lower than those in normal control group and AAV-shScramble group (F=257.408, 160.533;P＜0.05). The protein levels of Claudin-3, VEGF and Bcl-2 in AAV-shClaudin-3 group were lower than those in normal control group and AAV-shScramble group (F=129.671, 420.552, 62.669;P＜0.05), while the protein level of Caspase-3 in AAV-shClaudin-3 group was higher than that in normal control group and AAV-shScramble group (F=231.348,P＜0.05). Conclusion Down-regulation of Claudin-3 increases the expression of Caspase-3, reduces the expression of VEGF and Bcl-2, accelerates RGCs' apoptosis and inhibit the RGCs' axon growth.