CONTEXT: Vitamin D is increasingly recognized as an important immunomodulator. Lower levels of 25-hydroxyvitamin D (25[OH]D) and 1,25-dihydroxyvitamin D (1,25[OH]2D) are observed in persons living with HIV. OBJECTIVE: The purpose of this study was to evaluate the relationship of 25(OH)D, and 1,25(OH)2D to HIV viral load, and CD4+ T cells in HIV-infected adults. DESIGN: This was a cross-sectional study completed between January 2010 and April 2011. SETTING: This study was conducted with volunteers who received HIV care in Wisconsin at either a University-based HIV clinic or an urban community HIV clinic. PATIENTS: One hundred twelve adults with HIV infection participated in this study. MAIN OUTCOME MEASURES: The primary outcome for this study was the relationship between 1,25(OH)2D and HIV viral load. Secondary outcomes included relationships between 25(OH)D and HIV viral load, 25(OH)D and 1,25(OH)2D to CD4+ T cells, and predictors of vitamin D deficiency. RESULTS: The 112 volunteers included 24 women and 3 transgender individuals; 68% were from the university clinic, and 32% were from the urban clinic. Mean age was 44.2 years. The mean 25(OH)D level was 22.5 ng/mL; mean 1,25(OH)2D level was 23.5 pg/mL. Twenty-two percent had 25(OH)D ≤10 ng/mL; 53% had values <20 ng/mL, and 73% were ≤30 ng/mL. There was no association between vitamin D and CD4. A nonlinear relationship between viral load and 1,25(OH)2D was found. For 1,25(OH)2D below 32 pg/mL, for each 10 pg/mL decrease in 1,25(OH)2D, (log10) viral load increased by 0.84 (95% CI: 0.16-1.51, P = .015). For 1,25(OH)2D above 32 pg/mL, for each 10 pg/mL increase in 1,25(OH)2D, (log10) viral load increased by 0.36 (95% CI: 0.15-0.57, P = .0009). CONCLUSION: Vitamin D deficiency was common in this HIV population, as seen in other HIV cohorts. A novel, U-shaped relationship between 1,25(OH)2D and viral load, with the lowest and highest 1,25(OH)2D levels seen with high viral loads, was found and deserves further study.