Despite the critical role of vitamin B-12 in infant development, existing recommendations for infant and maternal intake during lactation are based on milk vitamin B-12 concentrations analyzed with outdated methods in a sample of 9 Brazilian women. Accurate quantification of vitamin B-12 in the milk matrix requires effective hydrolysis of the vitamin from haptocorrin, its binding protein. The objective of the present systematic review is to consider and critique evidence of associations between milk vitamin B-12 concentration and time postpartum, maternal vitamin B-12 consumption, maternal vitamin B-12 status, and sample collection methodology. A systematic search of published literature was undertaken using the US National Library of Medicine's MEDLINE/PubMed bibliographic search engine. Observational and intervention studies were included if research was original and vitamin B-12 concentration in human milk was measured using an appropriate method during the first 12 mo of lactation. Eleven studies met inclusion criteria. Vitamin B-12 concentration was highest in colostrum and decreased in a poorly delineated trajectory over the first 3-4 mo of lactation. There was some evidence of a positive association between habitual maternal vitamin B-12 intake and milk vitamin B-12 concentration in marginally nourished women. Supplementation with 50-250 µg vitamin B-12/d during pregnancy and lactation raised human milk vitamin B-12 concentrations while intervention was ongoing, whereas supplementation with 2.6-8.6 µg/d was effective in a population with poor baseline vitamin B-12 status but not in other populations. Whether milk vitamin B-12 concentration varies with maternal circulating vitamin B-12 concentrations or sampling methodology requires further research as existing data are conflicting. Additional research is needed to bridge knowledge gaps in the understanding of human milk vitamin B-12 concentrations. Reference values for vitamin B-12 in human milk and recommended intakes during infancy and lactation should be reevaluated using modern methods of analysis.