ABSTRACT Background Maintenance of high physical performance during aging might be supported by an adequate dietary intake of niacin, vitamins B-6 and B-12, and folate because these B vitamins are involved in multiple processes related to muscle functioning. However, not much is known about the association between dietary intake of these B vitamins and physical performance. Objectives The objectives of this study were to investigate the association between dietary intake of niacin, vitamins B-6 and B-12, and folate and physical performance in older adults and to explore mediation by niacin status and homocysteine concentrations. Methods We used baseline data from the New Dietary Strategies Addressing the Specific Needs of the Elderly Population for Healthy Aging in Europe (NU-AGE) trial, which included n = 1249 healthy older adults (aged 65–79 y) with complete data on dietary intake measured with 7-d food records and questionnaires on vitamin supplement use and physical performance measured with the short physical performance battery and handgrip dynamometry. Associations were assessed by adjusted linear mixed models. Results Intake of vitamin B-6 was related to lower chair rise test time [β: –0.033 ± 0.016 s (log); P = 0.043]. Vitamin B-6 intake was also significantly associated with handgrip strength, but for this association, a significant interaction effect between vitamin B-6 intake and physical activity level was found. In participants with the lowest level of physical activity, higher intake of vitamin B-6 tended to be associated with greater handgrip strength (β: 1.5 ± 0.8 kg; P = 0.051), whereas in participants in the highest quartile of physical activity, higher intake was associated with lower handgrip strength (β: –1.4 ± 0.7 kg; P = 0.041). No evidence was found for an association between intake of niacin, vitamin B-12, or folate and physical performance or for mediation by niacin status or homocysteine concentrations. Conclusions Vitamin B-6 intake was associated with better chair rise test time in a population of European healthy older adults and also with greater handgrip strength in participants with low physical activity only. Homocysteine concentrations did not mediate these associations. The NU-AGE trial was registered at clinicaltrials.gov as NCT01754012.