Objective To investigate the activity of glycogen synthase kinase-3β (GSK3β) on retinal ganglion cells (RGCs) under pressure, and the effect of GSK3β on the survival of RGCs.Methods This was an experimental study. Rat RGCs were mixed cultured in vitro and then divided into RGCs control group, RGCs pressure only group (60 mmHg of pressure cultivated for 24 hours),RGCs pressure group treated with 10 mmol/L lithium chloride (Licl, inhibitor of GSK3β) and RGCs pressure group treated with 20 mmol/L lithium chloride. Phospho-Ser9-GSK3β (pS9-GSK3β) was observed by immunocytochemical staining. Meanwhile, MTT method test optical density (OD) value to reflect the activity of RGCs of each group. The data for all groups were analyzed by an one-way ANOVA. Results Compared to the RGCs control group [mean optic density (MOD) was 0.085±0.023], the pressure only group expressed less pS9-GSK3β (0.057±0.012). However, both Licl treated groups, expressed more pS9-GSK3β (0.081±0.016 and 0.099±0.024) than the pressure only group.Meanwhile, as the concentration of Licl increased, the higher levels pS9-GSK3β were expressed. Moreover, MTT results showed that the difference of OD value in four groups had statistical significance (F=14.539, P＜0.05). OD value of pressure only group (0.109±0.016) was significantly lower than control group (0.135±).015) (P＜0.05), and RGCs viability was reduced. Compared to the OD value of the 20 mmol/L Licl treated group (0.159±0.014), the pressure only group showed lower OD value (P＜0.05), and the viability of RGCs increased. Conclusion The reduction of pS9-GSK3β in RGCs under pressure prompts GSK3β to be activated, so the viability of RGCs is lower than that of normal RGCs.However, Licl, as the inhibitor of GSK3β, maybe have the ability to protect RGCs from apoptosis.