The microbiome, the composite of bacteria present on the human body, has a critical role in the regulation of metabolism, immune function, and behavior. In pregnancy, the gut microbiota is altered, becoming less diverse as pregnancy progresses. The oral, vaginal, and placental microbiotas may also be affected by pregnancy. These changes may contribute to maternal physiological changes in pregnancy. Outside pregnancy, obesity, and diabetes are associated with changes to the composition of the gut microbiota. Whether or not obesity and diabetes compound pregnancy-associated changes to the microbiome is unclear. Manipulation of the gut microbiota by probiotic supplementation may be a strategy to prevent the development of gestational diabetes mellitus. In this chapter, the pregnancy-associated changes to the microbiome and the effects of probiotic supplementation on the prevention of gestational diabetes mellitus will be discussed.