Angiogenic VEGF isoforms are upregulated in diabetic retinopathy (DR), driving pathological growth and fluid leakage. Serine-Arginine-Rich protein kinase-1 (SRPK1) regulates VEGF splicing, and its inhibition blocks angiogenesis. We tested the hypothesis that SRPK1 is activated in diabetes, and an SRPK1 inhibitor (SPHINX31) switch VEGF splicing in DR and prevent increased vascular permeability into the retina. SRPK1 was activated by high glucose (HG), in a PKC-dependent manner, and was blocked by SPHINX31. HG induced release of SRSF1 from the nuclear speckles, which was also SRPK1 dependent and increased RPE monolayer admittance which was reversed by SRPK1 inhibition(p<0.05). Diabetes increased retinal permeability and thickness after 14 days which was blocked by treatment with SPHINX31 eye drops(p<0.0001). These results show that SRPK1 inhibition, administered as an eye drop, protected the retinal barrier from hyperglycaemia-associated loss of integrity in RPE cells in vitro and in diabetic rats in vivo. A clinical trial of another SRPK1 inhibitor has now been initiated in diabetic macular edema patients.