Vitamin D deficiency/insufficiency is currently considered to be a re-emerging public health problem globally. This study was designed to determine the prevalence of vitamin D deficiency and insufficiency and to investigate its trend from 2001 to 2013 in a longitudinal study of Iranian adults. This study was part of a population-based, longitudinal ongoing study of Iranian healthy adults aged 35 y and older at baseline. Serum vitamin D level was assessed in a sub-sample of 370 subjects, who were apparently healthy at the time of recruitment in 2001 and were free from MetS, in three phases (2001, 2007 and 2013) during the 12-y study period. Adjusted prevalence and trend of vitamin D deficiency were calculated. Mean serum vitamin D levels increased over the time of the study (52.12, 54.27 and 62.28 nmol/L, respectively) and the prevalence of vitamin D deficiency decreased (30.5, 27.0 and 24.4, respectively). However, the prevalence of vitamin D insufficiency did not change over this time period. The risk of vitamin D deficiency decreased significantly in 2007 [OR: 0.73 (95% CI: 0.53, 0.99)] and 2013 [OR: 0.50 (95% CI: 0.36, 0.70)] compared to the baseline. The present study demonstrated some improvement in serum vitamin D levels, while the prevalence of vitamin D inadequacy was still high. Considering the possible health consequences of vitamin D deficiency, there is an urgent need for developing population-wide strategies, such as supplementation and fortification, to prevent or control vitamin D deficiency.