We aimed to investigate whether health-related quality of life (HRQoL) is associated with retinal vascular caliber, an indicator of subclinical cardiovascular disease risk. 1600 students aged between 11-19 years (821 girls and 779 boys) were examined during 2009-2011. Retinal vessel caliber was measured from digital retinal images. HRQoL was assessed by the Pediatric Quality of Life Inventory (PedsQL). In the overall cohort, each 1-unit increase in PedsQL total score and the psychosocial summary score was associated with ~0.05 μm narrowing in retinal arteriolar caliber (multivariable-adjusted p-value = 0.01). Participants in the lowest versus highest tertile of PedsQL total score, psychosocial summary, social and school item scores had significantly wider retinal arteriolar caliber: 161.7 μm versus 160.2 μm (p = 0.02); 161.6 μm versus 160.0 μm (p = 0.02); 161.6 μm versus 159.9 μm (p = 0.002); and 161.6 μm versus 159.9 μm (p = 0.01), respectively. Significant interactions (p < 0.05) were observed between gender and PedsQL total score with retinal arteriolar calibre. In boys, inverse associations were observed between PedsQL total score (p = 0.01), psychosocial summary (p = 0.01), and social scores (p = 0.01) and retinal arteriolar caliber. No significant associations were observed between PedsQL scores and retinal vessel caliber in girls. Diminished HRQoL in adolescents was independently associated with structural retinal microvascular changes.