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Emerging evidence indicates that vitamin D has anti-oxidative properties. The present study investigates whether serum 25-hydroxyvitamin D [25(OH)D] is associated with biomarkers of anti-oxidative status in community-dwelling older adults using cross-sectional and longitudinal data.A total of 302 subjects aged 62 to 92 years from Germany (50.6°N) were analysed via cross-sectional approach. For longitudinal analysis, data of 153 subjects were available. Fasting blood samples from 2004 and 2012 were analysed for 25(OH)D concentrations, total anti-oxidative status (TAOS) as well as anti-oxidative enzymes, such as catalase (CAT), glutathione peroxidase (GPx) and superoxide dismutase (SOD). Multiple regression analyses were performed to examine the associations between 25(OH)D and parameters of anti-oxidative status.In cross-sectional analyses, 25(OH)D was a significant predictor of CAT (β = −0.166; P = 0.010), lg10 GPx (β = 0.136; P = 0.037) and TAOS (β = 0.121; P = 0.048) after adjusting for age, sex, percentage total body fat (TBF), month of blood sampling, smoking behaviour and use of vitamin D supplements. Longitudinal change in 25(OH)D concentration positively predicted change in TAOS (β = 0.224; P = 0.006) after adjusting for sex, baseline TAOS, age, smoking behaviour, use of vitamin D supplements and change in TBF, physical activity level, current time spent outdoors and dietary vitamin D intake.The maintenance of an adequate vitamin D status may have a beneficial impact on the anti-oxidative defence system in older adults on a long-term perspective.Longitudinal association of vitamin D with anti-oxidative status was investigated.Cross-sectional, vitamin D status was negatively associated with catalase activity.Longitudinal, vitamin D status predicted total anti-oxidative status in older adults.Maintenance of adequate vitamin D status may positively impact antioxidants.