Background Diabetic retinopathy (DR) is characterized by a gradually progressive alteration in the retinal microvasculature that leads to middle-aged adult acquired persistent blindness. Limited research has been conducted on DR pathogenesis at the gene level. Thus, we aimed to reveal novel key genes that might be associated with DR formation via a bioinformatics analysis. Methods The GSE53257 dataset from the Gene Expression Omnibus was downloaded for gene co-expression analysis. We identified significant gene modules via the Weighted Gene Co-expression Network Analysis, which was conducted by the Protein-Protein Interaction (PPI) Network via Cytoscape and from this we screened for key genes and gene sets for particular functional and pathway-specific enrichments. The hub gene expression was verified by real-time PCR in DR rats modeling and an external database. Results Two significant gene modules were identified. Significant key genes were predominantly associated with mitochondrial function, fatty acid oxidation and oxidative stress. Among all key genes analyzed, six up-regulated genes (i.e., SLC25A33, NDUFS1, MRPS23, CYB5R1, MECR, and MRPL15) were highly and significantly relevant in the context of DR formation. The PCR results showed that SLC25A33 and NDUFS1 expression were increased in DR rats modeling group. Conclusion Gene co-expression network analysis highlights the importance of mitochondria and oxidative stress in the pathophysiology of DR. DR co-expressing gene module was constructed and key genes were identified, and both SLC25A33 and NDUFS1 may serve as potential biomarker and therapeutic target for DR.