AbstractVitamin B₁₂, folate and homocysteine have long been implicated in mental illness, and growing evidence suggests that they may play a role in positive mental health. Elucidation of these relationships is confounded due to the dependence of homocysteine on available levels of vitamin B₁₂ and folate. Cross-sectional and longitudinal relationships between vitamin B₁₂, folate, homocysteine and subjective well-being were assessed in a sample of 391 older, community-living adults without clinically diagnosed depression. Levels of vitamin B₁₂, but not folate, influenced homocysteine levels 18 months later. Vitamin B₁₂, folate and their interaction significantly predicted levels of positive affect (PA) 18 months later, but had no impact on the levels of negative affect or life satisfaction. Cross-sectional relationships between homocysteine and PA were completely attenuated in the longitudinal analyses, suggesting that the cross-sectional relationship is driven by the dependence of homocysteine on vitamin B₁₂ and folate. This is the first study to offer some evidence of a causal link between levels of folate and vitamin B₁₂ on PA in a large, non-clinical population.