Diabetic retinopathy (DR) is a common complication of diabetes mellitus, characterized by progressive neurodegeneration and vision impairment. The Ca2+/calmodulin-dependent protein kinase II alpha (CaMK2A) and cAMP response element-binding protein (CREB) signaling pathway has been implicated in various neurological disorders. However, its role in DR pathogenesis remains elusive. We established a DR mouse model by streptozotocin administration and performed histological, biochemical, and molecular analyses to investigate the involvement of CaMK2A/CREB signaling and its interplay with mitophagy. Additionally, we employed in vitro high-glucose (HG) treatment in primary mouse retinal ganglion cells to dissect the underlying mechanisms. Pharmacological and genetic modulations were utilized to target CaMK2A/CREB pathway and mitophagy. In the DR model, we observed retinal degeneration, increased apoptosis, and reduced neurotransmitter production, accompanied by enhanced mitophagy and activation of the CaMK2A/CREB pathway. HG induction in retinal ganglion cells recapitulated these findings, and autophagy inhibition partially rescued cell death but failed to suppress CaMK2A/CREB activation, suggesting mitophagy as a downstream consequence. CaMK2A knockdown or CREB phosphorylation inhibition attenuated HG-induced mitophagy, apoptosis, and neurotransmitter depletion, while CREB activation exacerbated these effects. CaMK2A silencing mitigated DR progression, oxidative stress, inflammation, and neuronal loss, akin to dopamine/carbidopa administration in DR mouse model. Our findings reveal the involvement of CaMK2A/CREB signaling activation and enhanced mitophagy in DR, suggesting these pathways may be therapeutically relevant targets for DR management.