BACKGROUND The effect of vitamin B intake on physical function is not well known. OBJECTIVE To examine the prospective association of the intake of vitamins B6, B12 and folate with physical function impairment in older adults. METHODS We performed a prospective cohort study with 1630 participants from the Seniors-ENRICA study, a cohort of community-dwelling adults aged ≥60 years who were free of physical function impairment at baseline. In 2008-2010, nutrient intake was obtained through a validated computer-assisted face-to-face diet history. Study participants were followed-up through 2012 to assess incident impairment in agility and mobility, as well as impairment in overall physical functioning, defined as a decrease in the physical component summary of the 12-Item Short-Form Health Survey. RESULTS Over a median follow-up of 3.5 years, we identified 343 individuals with agility limitation, 212 with mobility limitation, and 457 with decreased overall physical functioning. A significant association was observed between intake of vitamin B6 and lower risk of impaired mobility (odds ratio [OR] for highest vs. lowest tertile: 0.66; 95% confidence interval [CI]:0.44-0.99; p-trend = 0.05). The results lost significance when additionally adjusted for vitamin B12 and folate, however the OR did not materially change. A higher consumption of important sources of vitamin B6, such as fish or fruit, was also related to a lower risk of impaired mobility (OR 100-g increase in fish: 0.50; 95% CI: 0.32-0.79; OR 100-g increase in fruit: 0.92; 95% CI: 0.84-1.01). No association was found between vitamin B12 and folate intake and physical function. CONCLUSIONS A higher intake of vitamin B6 and of several of its main sources, such as fish and fruit, was associated with lower risk of impaired mobility in Spanish older adults.