Background Influenza causes significant morbidity and mortality; the pandemic in 2009–2010 was a reminder of the potential for novel strains and antigenic changes. Studies have shown that vitamin D deficiency may be associated with poor vaccine immunogenicity, therefore we sought to determine if there was a correlation between 25-hydroxyvitamin D [25(OH)D] and influenza vaccine response. Methods A retrospective observational study was conducted among young, healthy military members to evaluate the association between total 25(OH)D levels with post influenza vaccination antibody titers and healthcare encounters during the 2009–10 influenza season. Univariate analyses were performed to evaluate whether 25(OH)D levels are associated with baseline characteristics and post-vaccination antibody responses. Multivariable logistic regression models were utilized to determine the associations between antibody responses and 25 (OH)D levels adjusting for possible confounders. Results A total of 437 subjects were studied. Most participants were young adults (91% were 18–39 years of age), 50% were male, and 56% resided in the southern U.S. Overall, 152 (34.8%) were vitamin D deficient, 167 (38.2%) insufficient, and 118 (27.0%) with normal 25(OH)D levels. There were no demographic differences by 25(OH)D category. Only 224 (51.3%) demonstrated a seroprotective anti-influenza post-vaccination titer, which did not vary by categorical 25(OH)D levels [vitamin D deficient vs. normal: OR 1.10 (0.68–1.78) and insufficient vs. normal: OR 1.25 (0.78–2.01)] or continuous vitamin D levels [OR 0.98 (0.84–1.15)]. There were also no associations with increased influenza like illnesses, respiratory diagnoses and healthcare encounters between the vitamin D groups. Conclusion Vitamin D insufficiency and deficiency were highly prevalent despite evaluating a young, healthy adult population. There were no significant associations between 25(OH)D levels and post-vaccination antibody titers to influenza vaccine. Further studies are required to discover strategies to improve vaccine efficacy as well as to determine the role of 25(OH)D in vaccine immunity.