The purpose of this study was to determine whether there was an association between serum 25-hydroxyvitamin D [25(OH)D] concentration and walking speed measured at usual and fast pace among older women. Usual- and fast-pace walking speeds and 25(OH)D concentrations were assessed in 739 randomized older women (mean age 80.2 ± 3.5 years) from the EPIDOS study. The following 25(OH)D thresholds were used: 10, 20, and 30 ng/mL. Walking speed was dichotomized on being in the worst quintile or not. Age, body mass index, number of chronic diseases, physical activity, quadriceps strength, cognition, use of psychoactive drugs, and serum parathyroid hormone were used as potential confounders. The results show that 90% of subjects had 25(OH)D insufficiency. Only fast-pace walking speed was significantly different between groups (p = .021) and decreased from normal serum 25(OH)D concentrations to severe insufficiency (trend p = .007). Serum 25(OH)D concentration was associated with walking speed at both usual and fast pace in the unadjusted linear regression (β = 0.16, p = .027 and β = 0.23, p = .009, respectively). This association remained significant only for fast-pace walking after adjustment (adjusted β = 0.18, p = .033) and was strengthened from a lower 25(OH)D value compared with usual pace [25(OH)D = 27.15 ng/mL for fast pace and 38.65 ng/mL for usual pace). Lastly, logistic regression showed a stronger association of serum 25(OH)D insufficiency with fast-pace walking speed whatever the 25(OH)D thresholds used [30 to 20 ng/mL: adjusted odds ratio (adjOR) = 6.01, p = .003; 20 to 10 ng/mL: adjOR = 4.10, p = .014; <10 ng/mL: adjOR = 6.95, p = .001) compared with usual pace (30 to 20 ng/mL: adjOR = 3.79, p = .022; 20 to 10 ng/mL: adjOR = 3.76, p = .016; <10 ng/mL: adjOR = 5.44, p = .003). The findings show a stronger positive association between 25(OH)D concentrations and fast-pace walking speed that is a more sensitive marker of neuromuscular functioning compared with usual-pace walking.