Background Immune activation plays a key role in HIV pathogenesis. Markers of inflammation have been associated with Vitamin D deficiency in the general population. Studies have also demonstrated associations of Vitamin D deficiency with increased risk of HIV progression and death. The relationship between persistent inflammation and immune activation during chronic HIV infection and Vitamin D deficiency remains unclear. Methods Cryopreserved specimens were analyzed from 663 participants at the time of enrollment from the Study to Understand the Natural History of HIV/AIDS in the Era of Effective Therapy (SUN Study) from 2004 to 2006. Biomarkers of inflammation, atherosclerosis, and coagulation were measured using enzyme-linked immunosorbent assays (ELISAs) and electrochemiluminescence. 25(OH)D, the stable precursor form of Vitamin D, was measured using a radioimmunoassay with levels defined as: normal (≥30ng/mL), insufficient (20-29 ng/mL) and deficient (<20 ng/mL). Monocyte phenotypes were assessed by flow cytometry. Linear and logistic regression models were used to determine statistical associations between biomarkers and Vitamin D deficiency. Results 25(OH)D levels were deficient in 251 (38%) participants, insufficient in 222 (34%), and normal in 190 (29%). Patients with Vitamin D deficiency, when compared to those with insufficient or normal Vitamin D levels, had increased levels of IL-6 (23%; p<0.01), TNF-α (21%, p = 0.03), D-dimer (24%, p = 0.01), higher proportions of CD14dimCD16+ (22%, p<0.01) and CX3CR1+ monocytes (48%; p<0.001) and decreased frequency of CCR2+ monocytes (-3.4%, p<0.001). In fully adjusted models, Vitamin D associations with abnormal biomarker levels persisted for IL-6 levels and CX3CR1+ and CCR2+ phenotypes. Conclusions Vitamin D deficiency is associated with greater inflammation and activated monocyte phenotypes. The role of Vitamin D deficiency in persistent immune activation and associated complications during chronic HIV disease should be further evaluated as a possible target for intervention.