Hyperhomocysteinemia has been implicated in visual dysfunction. We reported recently that mice with endogenous hyperhomocysteinemia, due to mutation of the cystathionine-β-synthase (cbs) gene, demonstrate loss of neurons in the retinal ganglion cell (RGC) layer and other retinal layers as homocysteine levels increase. Some clinical studies implicate hyperhomocysteinemia in the pathogenesis of diabetic retinopathy, which is also characterized by RGC loss. The present study used cbs(+/-) mice to determine whether modest elevation of plasma homocysteine, in the presence of diabetes, accelerates neuronal cell loss. Diabetes (DB) was induced in 3 wk old cbs(+/-) and wildtype mice using streptozotocin; four groups of mice were studied: DB cbs(+/-); non-DB cbs(+/-); DB cbs(+/+); non-DB cbs(+/+). One group of diabetic cbs(+/-) mice was maintained on a high methionine diet (HMD, 0.5% methionine drinking water) to increase plasma homocysteine slightly. Eyes were harvested at 5, 10 and 15 weeks post-onset of diabetes; retinal cryosections were examined by light microscopy and subjected to systematic morphometric analysis. Diabetic cbs(+/-) had significantly fewer RGCs at 5 weeks compared to age-matched, non-diabetic cbs(+/-) and wildtype controls (10.0 ± 0.5 versus 14.9 ± 0.5 and 15.8 ± 0.6 cells/100 µm retina length, respectively). Significant differences in retinas of DB/high homocysteine versus controls were obtained 15 wks post-onset of diabetes including fewer RGCS and decreased thickness of inner nuclear and plexiform layers. Moderate increases in plasma homocysteine coupled with diabetes cause a more dramatic alteration of retinal phenotype than elevated homocysteine or diabetes alone and suggest that diabetes accelerates the retinal neuronal death in hyperhomocysteinemic mice.