Objective To investigate the effect of recombinant human erythropoietin (rhEPO) on the expression of glutamine synthetase (GS) of cultured rat retinal Müller cells in high glucose environment in vitro. Methods Müller cells were isolated from retinas of 10 Sprague-Dawley rats at postnatal day three to five by trypsin digestion, and were randomly divided into six groups, including normal control group, high glucose group, high glucose +5 U/ml rhEPO group, high glucose+10 U/ml rhEPO group, high glucose +20 U/ml rhEPO group, high glucose+40 U/ml rhEPO groups. After 48 hours, the apoptosis of retinal Müller cells were assayed by terminal transferase-mediated DNA end labelling assay, and the expression levels of GS protein were detected with semi-quantitative immunocytochemistry. Results Compared with the normal control group, the cell viability and GS protein were reduced while the cell death increased in Müller cells cultured in high glucose, the difference was statistically significant (t=27.4,P＜0.01). Compared with the high glucose group, rhEPO treatment reduced the apoptotic Müller cells (t=857.2, 2 374.6, 2 473.2, 2 537.7;P＜0.01), induced the expression of GS proteins (t=3.2, 18.0, 22.5, 26.4; P＜0.05). Conclusions rhEPO can protect Müller cells from apoptosis under high glucose condition. The mechanism may be related to its function to up-regulate the GS protein expression, promote glutamic acid cycle, and reduce the excitotoxicity effects of high concentration of glutamate.