Diabetic retinopathy (DR) is the leading cause of blindness in diabetic patients, in which high glucose (HG)-induced Müller cell activation constitutes a central pathological event. This study aimed to untangle the critical role and mechanism of mitochondrial fission in this process. We found that under HG conditions, the level of p-Drp1 was significantly elevated (P<0.05), driving excessive mitochondrial fission. Functional experiments confirmed that artificially enhancing mitochondrial fission directly inhibited the Hippo signaling pathway (levels of core proteins p-MST1/2, p-LATS1, and p-YAP decreased, P<0.05, and YAP translocated to the nucleus), thereby activating Müller cells (expression of marker proteins GS and Kir4.1 decreased, while expression of GFAP, AQP4, and inflammatory mediators IL-1β, IL-6, VEGF increased, P<0.05). Key rescue experiments demonstrated that Drp1 silencing (reduced p-Drp1 level, P<0.05) reversed the aforementioned activation; however, co-administration of the Hippo pathway inhibitor XMU-MP-1 re-induced cell activation, proving that the Hippo pathway is a necessary downstream mediator of mitochondrial fission. In a diabetic rat model, elevated p-Drp1, Hippo pathway inhibition, and cell activation were similarly observed; the mitochondrial fission inhibitor Mdivi-1 alleviated this pathological process, whereas XMU-MP-1 counteracted its protective effects. This study systematically elucidates, from ex vivo to in vivo, the causal regulatory axis of "HG- mitochondrial fission- Hippo pathway inhibition—Müller cell activation," providing experimental evidence and a potential target for developing DR-targeted therapeutic strategies centered on intervening in mitochondrial dynamics.