ABSTRACTThe microbiota ofDrosophila melanogasterhas a substantial impact on host physiology and nutrition. Some effects may involve vitamin provisioning, but the relationships between microbe-derived vitamins, diet, and host health remain to be established systematically. We explored the contribution of microbiota in supplying sufficient dietary thiamine (vitamin B1) to supportD. melanogasterat different stages of its life cycle. Using chemically defined diets with different levels of available thiamine, we found that the interaction of thiamine concentration and microbiota did not affect the longevity of adultD. melanogaster. Likewise, this interplay did not have an impact on egg production. However, we determined that thiamine availability has a large impact on offspring development, as axenic offspring were unable to develop on a thiamine-free diet. Offspring survived on the diet only when the microbiota was present or added back, demonstrating that the microbiota was able to provide enough thiamine to support host development. Through gnotobiotic studies, we determined thatAcetobacter pomorum, a common member of the microbiota, was able to rescue development of larvae raised on the no-thiamine diet. Further, it was the only microbiota member that produced measurable amounts of thiamine when grown on the thiamine-free fly medium. Its close relativeAcetobacter pasteurianusalso rescued larvae; however, a thiamine auxotrophic mutant strain was unable to support larval growth and development. The results demonstrate that theD. melanogastermicrobiota functions to provision thiamine to its host in a low-thiamine environment.IMPORTANCEThere has been a long-standing assumption that the microbiota of animals provides their hosts with essential B vitamins; however, there is not a wealth of empirical evidence supporting this idea, especially for vitamin B1(thiamine). To determine whether this assumption is true, we usedDrosophila melanogasterand chemically defined diets with different thiamine concentrations as a model. We found that the microbiota does provide thiamine to its host, enough to allow the development of flies on a thiamine-free diet. The power of theDrosophila-microbiota system allowed us to determine that one microbiota member in particular,Acetobacter pomorum, is responsible for the thiamine provisioning. Thereby, our study verifies this long-standing hypothesis. Finally, the methods used in this work are applicable for interrogating the underpinnings of other aspects of the tripartite interaction between diet, host, and microbiota.