With the improvement of living standards and the lack of nutrition awareness during pregnancy, the phenomenon of excessive weight gain (EWG) of pregnancy is increasing. EWG during pregnancy has profound effects on the health of mother and offspring. The role of intestinal flora in regulating metabolic diseases has gradually attracted attention in recent years. The study explored the effect of EWG during pregnancy on gut microbiota, and analyzed the diversity and composition of gut microbiota in pregnant women in third trimester. Fecal samples were collected and divided into: insufficient weight gain (IWG) during pregnancy (group A1, N = 4), and appropriate weight gain (AWG) during pregnancy (group A2, N = 9), and EWG during pregnancy (N = 9 in A3 group). MiSeq high-throughput sequencing technology and bioinformatics analysis were introduced to investigate relationship of gestational weight gain and maternal gut microbiota. General data analysis showed that gestational weight gain and delivery mode have significant differences among the three groups. The overall level and diversity of intestinal microbiota in A1 and A3 group were increased. Composition of gut microbiota has no difference among three groups at the phylum level, but species of gut microbiota were different. Alpha diversity index analysis showed that the richness of A3 group was increased versus A2 group. EWG during pregnancy affects the abundance and proportion of gut microbiota in the third trimester. Therefore, maintaining moderate weight gain during pregnancy helps to maintain intestinal homeostasis.