The objectives of this study were to investigate how diabetes mellitus (DM) influences responsiveness of retinal neovessels to lysophosphatidic acid (LPA) and to elucidate the underlying mechanism. To this end, we used an ex vivo assay in which neovessels sprouted from retinal explants (isolated from either control or DM mice) when cultured between two layers of collagen and in the presence of vascular endothelial growth factor-A. While DM had no effect on the formation of neovessels, it prevented LPA-induced regression. High-glucose (HG) treatment of retinal explants mimicked the DM phenotype. Similarly, primary retinal endothelial cells (RECs), which were subjected to HG treatment, organized into tubes that were resistant to LPA. HG caused LPA resistance within RECs by elevating ROS, which activated Src-family kinases that stimulated the extracellular signal-related kinase (Erk) pathway, which antagonized LPA-mediated signaling events that were required for regression. This ROS/Src/Erk pathway mechanism appeared to be the same route by which DM induced LPA resistance of retinal neovessels. We conclude that DM/HG reprograms signaling pathways in RECs to induce a state of LPA resistance.