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A systematic review and meta-analysis was undertaken to examine and quantify the effects of B vitamin supplementation on mood in both healthy and &#8216;at-risk&#8217; populations. A systematic search identified all available randomised controlled trials (RCTs) of daily supplementation with &#8805;3 B group vitamins with an intervention period of at least four weeks. Random effects models for a standardized mean difference were used to test for overall effect. Heterogeneity was tested using the I² statistic. Eighteen articles (16 trials, 2015 participants) were included, of which 12 were eligible for meta-analysis. Eleven of the 18 articles reported a positive effect for B vitamins over a placebo for overall mood or a facet of mood. Of the eight studies in &#8216;at-risk&#8217; cohorts, five found a significant benefit to mood. Regarding individual facets of mood, B vitamin supplementation benefited stress (<i>n</i> = 958, SMD = 0.23, 95% CI = 0.02, 0.45, <i>p</i> = 0.03). A benefit to depressive symptoms did not reach significance (<i>n</i> = 568, SMD = 0.15, 95% CI = &#8722;0.01, 0.32, <i>p</i> = 0.07), and there was no effect on anxiety (<i>n</i> = 562, SMD = 0.03, 95% CI = &#8722;0.13, 0.20, <i>p</i> = 0.71). The review provides evidence for the benefit of B vitamin supplementation in healthy and at-risk populations for stress, but not for depressive symptoms or anxiety. B vitamin supplementation may particularly benefit populations who are at risk due to (1) poor nutrient status or (2) poor mood status.