The effect of hypoxia on expression and function of L-glutamate/L-aspartate transporter (GLAST) and glutamine synthetase(GS) was investigated in mouse retinal Müller cells(RMCs). Mouse RMCs were cultured by enzymatic digestion method. RMCs cultures were treated with CoCl₂ (125 μmol/L) for 6 h, 12 h, 24 h, 48 h or 72 h respectively in vitro. RMCs cultures were maintained without CoCl₂ in normal control group. The expression of GLAST and GS was determined by RT-PCR, Western blotting and immunocytochemical staining. L-[3,4-³H]- glutamic acid uptake was used to quantify glutamate uptake function of RMCs. The apoptosis of RMCs was confirmed by annexin V-FITC/PI staining. In early-stage of CoCl₂-induced hypoxia (treated with CoCl₂ for 6 h, 12 h, 24 h or 48 h)，the expression of GLAST was up-regulated (P < 0.001) and reached the maximum when RMCs have been treated with CoCl₂ for 12 h (P < 0.05). After RMCs having been treated with CoCl₂ for 72 h, the expression of GLAST had no difference compared to normal control (P > 0.05). CoCl₂-induced hypoxia (treated with CoCl₂ for 6 h, 12 h, 24 h, 48 h or 72 h) also up-regulated the expression of GS (P < 0.001) which reached the maximum when RMCs have been treated with CoCl₂ for 48h (P < 0.001). The L-[3,4-³H]-glutamic acid uptake of hypoxia groups were the higher than normal control group (P < 0.05), and after having treated RMCs with CoCl2 for 48 h, the L-[3,4-³H]-glutamic acid uptake was the highest (P < 0.005). Treatments with CoCl₂ did not induce apoptosis in RMCs. In early hypoxia stage, GLAST and GS were up-regulated and extracellular glutamate uptake increased. However, continued hypoxia causes gradual dysfunction and reduction of GLAST and GS, as well as glutamate uptake.