The aim of the present study was to investigate glutamate homeostasis in retinal degeneration-induced changes and the potential mechanisms of glutamate-mediated excitotoxicity in a rat model. The expression of vesicular glutamate transporter-1 (VGLUT-1) and protein kinase Cα (PKCα) in wild-type and Royal College of Surgeons (RCS) rat retinas, at postnatal Day 15 (P15), P30, P60 and P90, were detected using quantitative real-time polymerase chain reaction and immunohistochemistry. The levels of glutamine synthetase (GS) and L-glutamate/L-aspartate transporter (GLAST) were evaluated by western blotting. Compared with wild-type rats, outer nuclear layer thickness was significantly thinner and VGLUT-1 expression was upregulated in a time-dependent pattern in RCS rats. The ratio of VGLUT-1 to PKCα in RCS rats peaked at P60 (p<0.01) and subsequently decreased by P90 (p<0.01), while it remained constant in wild-type rats. The expression of GS increased gradually from P30 to P90 in RCS rats (p<0.01), while it remained constant in wild-type rats at various time-points. No significant difference in GLAST expression was found between RCS and wild-type rats at all stages of retinal degeneration. Our results confirm the occurrence of glutamate-mediated excitotoxicity to RCS rat retinas and provide an experimental foundation for safeguarding the remnant visual function in retinal degenerative disorders.