ObjectiveTo assess whether higher 25-hydroxyvitamin D (25OHD) levels are associated with subsequent better immune responses to hepatitis B and Streptococcus pneumoniae vaccination in HIV-infected patients.Methods25OHD was measured on stored baseline plasma samples from two randomized vaccine trials in HIV-infected adults: the ANRS HB03 VIHVAC B trial and an immunological sub-study of the ANRS 114-PNEUMOVAC trial. In ANRS HB03 VIHVAC B, participants received three or four doses of recombinant HBV vaccine strategies. Anti-HBs IgG titers were measured four weeks after the last injection. Associations between baseline 25OHD levels and ordered IgG response categories were analyzed in multivariable proportional odds models. In the ANRS 114-PNEUMOVAC sub-study, two strategies of pneumococcal vaccination were tested, cellular immune responses were measured at repeated time points, and IgG responses four weeks after the last vaccine injection. Exploratory statistical analyses were performed on this sub-study data set.ResultsThree hundred and thirty-nine ANRS HB03 VIHVAC B and 25 ANRS 114-PNEUMOVAC sub-study participants were included in the analyses. Median age in each of the two studies was 43 years, 68% were male, and 77–92% on antiretroviral treatment. Median 25OHD level was 18 ng/mL (IQR: 12–25) and 24 ng/mL (IQR: 13–32) in the two trial populations, respectively. In the multivariable model, there was no significant association between baseline 25OHD level and vaccine responses in ANRS HB03 VIHVAC B (proportional odds ratio 0.83 per 10 ng/mL 25OHD increase; 95% confidence interval 0.65–1.07, p = 0.14). Exploratory analyses of ANRS 114-PNEUMOVAC showed consistent results.ConclusionThis study does not support a positive association between 25OHD and immune responses to hepatitis B or pneumococcal vaccination in HIV-infected patients.