BACKGROUND: Adverse neuromuscular events have been described in case of low serum 25-hydroxyvitamin D (25OHD) concentrations, suggesting that vitamin D may be involved in gait stability. The objective of this cross-sectional study was to examine the association between stride-to-stride variability of stride time (STV) and serum 25OHD concentration in adults aged 65 years and older. METHODS: STV and 25OHD concentration were assessed in 411 community-dwelling older adults (mean age 70.4 +/- 1.8 years, 57.9% women). The following established 25OHD thresholds were used: severe 25OHD insufficiency <10 ng/mL, moderate 10-30 ng/mL, and normal >30 ng/mL. Age, number of drugs used per day, use of psychoactive drugs, depressive symptoms, cognitive decline, history of falls, distance visual acuity, lower limb proprioception, center of mass (CoM) motion, and walking speed were considered as potential confounders. RESULTS: A total of 16.6% (n = 68) of subjects had severe 25OHD insufficiency, 70.3% (n = 289) moderate insufficiency, and 13.1% (n = 54) normal concentrations. In the full adjusted and the stepwise backward linear regression models, high STV (worse performance) was associated with severe 25OHD insufficiency (p = 0.028 and p = 0.044, respectively), high CoM motion (p = 0.031 and p = 0.014, respectively), and low lower limb proprioception score (p = 0.017 and p = 0.008, respectively). The stepwise backward regression model also showed that high STV was associated with female gender (p = 0.041). CONCLUSIONS: Low serum 25OHD concentrations were associated with high STV reflecting a disturbed gait control. This association could be explained by a possible action of vitamin D on different components involved in gait control.