Objective To investigate the relationship between the relative inner nuclear layer (rINL) and the development and progression of diabetic retinopathy (DR). Design Nested case-control study. Participants The study included 95 patients (95 eyes) with type 2 diabetes mellitus (T2DM) who visited Beijing Tongren Hospital, Capital Medical University, from 2019 to 2022. Among them, 31 patients (31 eyes) had no DR (DM group), 34 patients (34 eyes) had non-proliferative DR (NPDR), and 30 patients (30 eyes) had proliferative DR (PDR). Additionally, 35 age- and gender-matched healthy subjects (35 eyes) were enrolled as controls. Methods General clinical data, including gender, age, and duration of T2DM, were collected. Fasting blood samples were obtained for biochemical analysis, including glycated hemoglobin (HbA1c), fasting blood glucose (FBG), total cholesterol, triglycerides, low-density lipoprotein cholesterol, and high-density lipoprotein cholesterol (HDL-C). All participants underwent comprehensive ophthalmic examinations, including visual acuity, intraocular pressure, fundus photography, and swept-source optical coherence tomography (SS-OCT). Retinal images obtained via SS-OCT were segmented into separate zones. Full retinal thickness and INL thickness were measured for each zone, and the rINL (defined as INL thickness divided by total retinal thickness, multiplied by 100%) was calculated and subsequently analyzed. Main Outcome Measures rINI, NLL thickness, full retinal thickness, blood lipid metabolism indicators. Results Compared with the control group, the rINL was significantly lower in the PDR group (P=0.005). Furthermore, the outer ring rINL was significantly lower in the DM, NPDR, and PDR groups compared to the control group (P<0.001). Spearman correlation analysis revealed a significant negative correlation between the outer ring rINL and T2DM duration, HbA1c, and FBG levels (P<0.01). Univariate logistic regression analysis showed that a greater outer ring rINL was a protective factor against DR development (OR=0.578, 95% CI: 0.447~0.748, P<0.001). Multivariate ordered logistic regression analysis further confirmed that both outer ring rINL (OR=0.739, 95% CI: 0.558~0.978, P=0.034) and HDL-C (OR=0.054, 95% CI: 0.013~0.222, P<0.001) were independent protective factors against the development and progression of DR. Conclusion The outer ring rINL is an independent protective factor for the development and progression of DR, serves as a promissing novel imaging biomarker for DR diagnosis and management, and is associated with systemic lipid metabolism.