PURPOSE: To describe the relationship of retinal arteriolar and venular caliber with retinal nerve fiber layer (RNFL) thickness. METHODS: A population-based, cross-sectional study of Malay persons aged 40 to 80 years residing in Singapore was conducted from 2004 to 2006. Retinal arteriolar and venular calibers were measured with a computer-based technique according to a validated, standardized protocol. RNFL was assessed with retinal tomography. RESULTS: There were 2706 persons with gradable retinal photographs and HRT images of acceptable quality available for analysis. In linear regression models that adjusted for age, sex, diabetes, body mass index, intraocular pressure, and other factors, each standard deviation decrease in arteriolar and venular caliber was associated with a 5.81- and 8.37-microm decrease, respectively, in mean global RNFL thickness (both P < 0.001). These associations remained similar after persons with glaucoma were excluded. In persons with glaucoma, retinal venular caliber was independently associated with the temporal and temporal-to-inferior region of mean RNFL thickness in multivariate analysis; each SD decrease in retinal venular diameter was associated with an 8.54-microm decrease in the temporal region (P = 0.022), and a 38.32-microm decrease in the temporal-to-inferior region (P = 0.006) of mean RNFL thickness. CONCLUSIONS: Narrower retinal vessel caliber was associated with reduced RNFL thickness in this Asian population.