Work in multiple organs has suggested that toll-like receptor 4 (TLR4) may play a role in insulin resistance. Additional studies have shown a negative role for TLR4 on retinal health. We have previously reported that beta-adrenergic receptors can regulate both TLR4 signal transduction, as well as insulin signaling in the retina and in retinal endothelial cells. Thus, we hypothesized that TLR4 would regulate retinal insulin signaling.We used endothelial cell-specific TLR4 knockout mice, as well as TLR4-overexpressing mice for these studies.Western blotting and ELISA analyses were done for investigations of insulin receptor, insulin receptor substrate 1 (IRS-1) serine 307, and Akt phosphorylation, as well as cleaved caspase 3 levels in the mouse retina.We found that loss of TLR4 led to increased insulin receptor and Akt phosphorylation, as well as decreased IRS-1(Ser307) levels. In support of these results, TLR4 overexpression decreased insulin signaling and the cleavage of caspase 3.Therefore, these results suggest that TLR4 plays a key role in insulin signaling in the retina. Reduction of TLR4 levels may be protective to the retina.