Shifts in the maternal gut microbiota have been implicated in the development of gestational diabetes mellitus (GDM). Understanding the interaction between gut microbiota and host glucose metabolism will provide a new target of prediction and treatment. In this nested case-control study, we aimed to investigate the causal effects of gut microbiota from GDM patients on the glucose metabolism of germ-free (GF) mice. Stool and peripheral blood samples, as well as clinical information, were collected from 45 GDM patients and 45 healthy controls (matched by age and prepregnancy body mass index (BMI)) in the first and second trimester. Gut microbiota profiles were explored by next-generation sequencing of the 16S rRNA gene, and inflammatory factors in peripheral blood were analyzed by enzyme-linked immunosorbent assay. Fecal samples from GDM and non-GDM donors were transferred to GF mice. The gut microbiota of women with GDM showed reduced richness, specifically decreased Bacteroides and Akkermansia, as well as increased Faecalibacterium. The relative abundance of Akkermansia was negatively associated with blood glucose levels, and the relative abundance of Faecalibacterium was positively related to inflammatory factor concentrations. The transfer of fecal microbiota from GDM and non-GDM donors to GF mice resulted in different gut microbiota colonization patterns, and hyperglycemia was induced in mice that received GDM donor microbiota. These results suggested that the shifting pattern of gut microbiota in GDM patients contributed to disease pathogenesis.