Background/Objectives:The roles of vitamin B12 and homocysteine concentration in depression are not clear. We investigated cross-sectional and prospective associations of serum vitamin B12 and plasma homocysteine with depressive symptoms in Dutch older adults.Subjects/Methods:In the Longitudinal Aging Study Amsterdam (LASA), blood was collected in 1995/1996 among 1352 men and women aged ≥65 years. Depressive symptoms were assessed with the Center for Epidemiologic Studies Depression Scale (CES-D) six times from 1995/1996 to 2011/2012. Multiple linear regression and mixed models were used to assess whether vitamin B12 and homocysteine were associated with severity at baseline and course of depressive symptoms over 16 years. Cox regression analyses were performed for the associations with incidence of depression (CES-D ≥16 and/or antidepressant use). All analyses were adjusted for sociodemographic characteristics and lifestyle factors.Results:Vitamin B12 was neither cross-sectionally (n=1205) nor prospectively (n=1012) associated with depressive symptoms (adjusted β for CES-D over time, lowest versus highest quartile: -0.04 (95% confidence interval (CI): -0.15-0.06)). We also found no association with incident depression (n=853), except for a higher risk of depression over time in younger participants (aged 64.8-73.4 years; continuous vitamin B12, adjusted hazard ratio per s.d.: 1.38 (95% CI: 1.10-1.72)). For homocysteine, no associations were found, except for a lower risk of depression in younger participants.Conclusions:Our study did not confirm earlier shown associations of serum vitamin B12 and plasma homocysteine with severity and course of depressive symptoms and incidence of depression in older adults. Further research into the influence of homocysteine metabolism on mental health is needed.