BACKGROUND: Beyond its role in calcium homeostasis, vitamin D plays a critical role in immunological responses to pathogens. We evaluated the relationship between 25-OH vitamin D levels and susceptibility to natural H1N1 infection and H1N1 vaccine responses in HIV infected individuals. METHODS: This was a sub study of an H1N1 vaccine trial conducted at the University of Pennsylvania in 2009/10. We compared the 25-OH vitamin D levels among individuals with and without baseline evidence of prior H1N1 infection and between vaccine responders and non-responders. RESULTS: 120 participants enrolled in the trial, 71% male, 68% African American, median age 46 years. The majority had controlled HIV disease. At baseline, 86% had 25-OH vitamin D levels < 30 ng/ml and 54% had levels < 20 ng/ml. Thirty participants (25%) had evidence of prior H1N1 exposure. There was no difference in mean 25-OH vitamin D levels among patients with or without prior natural H1N1 infection (21 ng/ml vs 20 ng/ml, p=0.72). Among participants without previous H1N1 exposure, only 61% developed protective antibody titers following vaccination. 25-OH vitamin D levels were similar between vaccine responders (20 ng/ml) and non-responders (20 ng/ml) (p=0.83). CONCLUSION: Although 25-OH vitamin D deficiency was very common among HIV-infected individuals, it was not associated with natural susceptibility to H1N1 or to vaccine responses.