BACKGROUND: Vitamin D is an important determinant of bone health and also plays a major role in the regulation of the immune system. Interestingly, vitamin D status before the start of highly active antiretroviral therapy (HAART) has been recently associated with HIV disease progression and overall mortality in HIV-positive pregnant women. We prospectively studied vitamin D status in HIV individuals on HAART in Belgium. We selected samples from HIV-positive adults starting HAART with a pre-HAART CD4 T-cell count >100 cells/mm3 followed up for at least 12 months without a treatment change. We compared 25-hydroxyvitamin D plasma [25-(OH)D] concentration in paired samples before and after 12 months of HAART. 25-(OH)D levels are presented using two different cut-offs: <20 ng/ml and <30 ng/ml. RESULTS: Vitamin D deficiency was common before HAART, the frequency of plasma 25-(OH)D concentrations below 20 ng/ml and 30 below ng/ml was 43.7% and 70.1% respectively. After 12 months on HAART, the frequency increased to 47.1% and 81.6%. HAART for 12 months was associated with a significant decrease of plasma 25-(OH)D concentration (p=0.001). Decreasing plasma 25-(OH)D concentration on HAART was associated in the multivariate model with NNRTI-based regimen (p=0.001) and lower body weight (p=0.008). Plasma 25-(OH)D concentrations decreased significantly in both nevirapine and efavirenz-containing regimens but not in PI-treated patients. CONCLUSIONS: Vitamin D deficiency is frequent in HIV-positive individuals and NNRTI therapy further decreases 25-(OH)D concentrations. Consequently, vitamin D status need to be checked regularly in all HIV-infected patients and vitamin D supplementation should be given when needed.