Purpose: To investigate ocular and systemic factors associated with the retinal arterial wall-to-lumen ratio (WLR) and to determine the relative contribution of genetic and environmental variation to WLR in healthy adults. Methods: This cross-sectional twin study included 78 monozygotic and 67 dizygotic same-sex twin pairs aged 58.4 ± 9.8 (mean ± SD) years. Lumen diameter (LD) and outer diameter (OD) of a superotemporal retinal artery were measured using adaptive optics fundus photography, and the WLR was calculated. Linear mixed model regression analysis of associations with WLR comprised the descriptive variables ocular axial length (AL), intraocular pressure (IOP), height, weight, body mass index (BMI), smoking, blood pressure, high density (HDL), low density (LDL) and very low density (VLDL) lipoproteins, total cholesterol and triglycerides. The relative influence of genes and environment on WLR was calculated through polygenetic modelling. Results: Increasing age and arterial blood pressure were associated with a higher WLR, while increasing retinal artery OD and ocular AL were associated with a lower WLR. Sex, smoking status, BMI, IOP, cholesterol levels or triglycerides had no detectable impact on the WLR. Broad-sense heritability of WLR was 21% (95% CI: 1–41%), while environmental factors accounted for the remaining 79% of the interindividual variance (95% CI: 59–99%). Conclusion: Retinal artery wall thickness was closely linked to increasing age and higher arterial blood pressure, the latter being mediated by the environment over genes.