Vitamin D is necessary to maintain healthy bones, and may prevent other chronic diseases. There is limited information regarding the vitamin D status of people living in climates with relatively high ambient ultraviolet radiation. We therefore aimed to determine serum 25(OH)D levels in a group of office-workers in subtropical Australia. We collected blood from 129 office workers in summer (n = 129) and 175 in winter (91 in both seasons). Serum 25(OH)D was estimated using a commercial chemiluminescent immunoassay and we asked participants to complete questionnaires about sun exposure and diet for the month prior to blood collection. Summer and winter mean serum 25(OH)D was 74 (95% CI 70–77) nmol L−1 and 54 (95% CI 51–57) nmol L−1, respectively. In summer, 14% of participants were classed as “insufficient,” compared with 51% in winter. High 25(OH)D levels in summer were associated with time spent outdoors in nonpeak UV periods, while in winter high levels were associated with intake of vitamin D from food or supplements. The high prevalence of vitamin D insufficiency observed in this population highlights the need for further examination of the relation between sunlight and vitamin D production to enable more accurate sun exposure recommendations.