Background/Objective: We aimed to describe serum 25-hydroxyvitamin D (25OHD) concentrations in older Europeans and to investigate associations between 25OHD and lifestyle factors, including dietary intake and supplement use. Subjects/Methods: Men and women aged ≥65 years were recruited from seven centres across north to south Europe. Serum 25OHD2 and 25OHD3 concentrations were measured by liquid chromatography tandem mass spectrometry (LC-MS/MS) in 4495 samples and total 25OHD (25OHD2 + 25OHD3) was adjusted for season of blood collection. Results: The mean (25th, 75th quartile) of seasonally adjusted 25OHD was 46 (34, 65) nmol/L, with the highest concentration of 25OHD in Bergen [61 (49, 79) nmol/L], and the lowest in Paris [36 (24, 57) nmol/L)]. Vitamin D deficiency (25-50 nmol/L) and vitamin D insufficiency (50-75 nmol/L) were found in 41% and 33% of the population, respectively. In multivariable analysis controlled for confounders, seasonally adjusted 25OHD concentrations were significantly (p<0.05) lower in smokers and participants with self-reported diabetes and higher with increasing dietary vitamin D, and supplement use with fish liver oil, omega-3 and vitamin D. Additionally in further analysis excluding Bergen, 25OHD was associated with higher intakes of oily fish and increasing UVB exposure. We observed low concentrations of 25OHD in older people in Europe. Conclusions: Our findings of the higher 25OHD concentrations in supplement users (omega-3 fish oil, fish liver oil, vitamin D) add to current recommendations to reduce vitamin D deficiency. We were unable to fully assess the role of dietary vitamin D as we lacked information on vitamin D fortified foods.