OBJECTIVES: This study aimed to assess the independent associations of serum levels of vitamin B12 and plasma concentrations of homocysteine with gait speed decline. DESIGN, SETTING, PARTICIPANTS: This study utilized longitudinal analysis of participants 50 years or older from The Baltimore Longitudinal Study of Aging, N=774. MEASUREMENTS: Gait speed (m/s) was assessed using the 6-meter usual pace test. Vitamin B12 and homocysteine concentrations were collected using standard clinical protocols. Linear mixed effects regression was stratified by baseline age category (50-69, 70-79, and ≥80 years old). RESULTS: Mean follow-up time for the total study sample was 5.4 ± 2.0 years. No association between vitamin B12 and gait speed decline over the follow-up time for any age group was found. Elevated homocysteine concentrations were associated with decline in gait speed after adjustment for covariates (50-69: β= -0.005, p=.057; 70-79: β= -0.013, p<.001, ≥80: β= -0.007, p=.054). CONCLUSION: Homocysteine and vitamin B12 are inversely related, yet only homocysteine was associated with gait speed decline in this population of healthy older adults. Given these results, future research should be directed towards investigating the relationship in populations with greater variation in vitamin B12 concentrations and other mechanisms influencing homocysteine concentrations.