Background <p>Vitamin A deficiency remains a clinically relevant nutritional issue, yet data on real-world testing patterns and vitamin A status in Korea are limited.</p> Methods <p>We conducted a retrospective observational study analyzing serum vitamin A (retinol) test results performed for diagnostic purposes at a large referral laboratory in Korea between 2016 and 2024. Vitamin A deficiency (&lt; 0.70 µmol/L) and testing patterns were evaluated by age, sex, calendar year, and referring institution type, and factors associated with deficiency were examined using multivariable logistic regression; population-level prevalence was additionally assessed using weighted data from KNHANES 2016–2018.</p> Results <p>A total of 15,042 serum vitamin A measurements from 9,608 individuals were analyzed. The overall prevalence of vitamin A deficiency was 7.6%. In the GC Labs population, vitamin A deficiency showed a U-shaped age distribution, with higher prevalence in younger age groups and in older adults, particularly those aged ≥ 80 years, who had the highest risk (adjusted odds ratio 9.52). Male sex and testing at public health centers were also independently associated with deficiency. In the KNHANES 2016–2018 population, the weighted prevalence of vitamin A deficiency was low (0.76%); however, a similar U-shaped age distribution was observed, with relatively higher prevalence in adolescents and older adults.</p> Conclusions <p>Although vitamin A deficiency does not represent a major public health burden in Korea, older adults and specific high-risk groups may be vulnerable. These findings provide a real-world overview of vitamin A testing practices and support targeted clinical and nutritional assessment in selected populations.</p>