Little is known about the effect of working conditions on vitamin D status in Southeast Asia, where vitamin D deficiency is common despite the presence of sunlight all year round in most places. We examined the prevalence of vitamin D deficiency and its associated work-related factors among indoor workers using the data of 213 participants (aged ≥21 years) from a workplace cohort study in Singapore. Vitamin D deficiency was defined as serum 25-hydroxyvitamin D (25(OH)D) concentration <50 nmol/L. Data on work-related factors, socio-demographic characteristics, and lifestyle habits were collected using standardized questionnaires. Clinical and biochemical measurements were performed using standard tools and protocols. Multivariate Poisson regression was used to examine the independent association of work-related factors with vitamin D deficiency. Mean serum 25(OH)D concentration was 59.6 nmol/L. The prevalence of vitamin D deficiency was 32.9% (95% confidence interval (CI): 26.6–39.6%). In the multivariate analysis, office workers (prevalence ratio (PR): 2.16, 95% CI: 1.12–4.16 versus control room workers), workshop workers (PR: 2.25, 95% CI: 1.05–4.81 versus control room workers), and night shift workers (PR: 1.31, 95% CI: 1.03–1.67) were at a greater risk for vitamin D deficiency. Workplace policies and wellness programs should encourage workers to take regular breaks to go outdoors for sunlight exposure and to consume adequate amounts of vitamin D-rich foods to maintain optimal vitamin D levels.