The community structure of gut microbiota changes during pregnancy, which also affects the synthesis of short-chain fatty acids (SCFAs). However, the distribution of gut microbiota composition and metabolite SCFA levels are poorly understood in women undergoing assisted reproductive technology (ART). To evaluate the changes in gut microbiota composition and metabolic SCFAs in women who received assisted reproduction treatment. Sixty-three pregnant women with spontaneous pregnancy (SP) and nine with ART pregnancy were recruited to provide fecal samples. Gut microbiota abundance and SCFA levels were determined by 16S ribosomal RNA (rRNA) gene amplicon sequencing and gas chromatography-mass spectrometry (GC-MS). The ART group showed decreased alpha diversity (the species richness or evenness in a sample). The principal coordinates analysis (a method of analysing beta diversity) showed significant difference in gut microbiota between the ART group versus the SP group (unweighted UniFrac distance, R2=0.04, P=0.003). Proteobacteria, Blautia and Escherichia-Shigella were enriched in the ART group, whereas the relative abundance of beneficial intestinal bacteria Faecalibacterium was lower than in the SP group. Different modes of conception were associated with several SCFAs (valeric acid (r=-0.280; P=0.017); isocaproic acid (r=-0.330; P=0.005); caproic acid (r=-0.336; P=0.004)). Significantly different SCFAs between the two groups were synchronously associated with the differential gut microbiota. The diversity and abundance of gut microbiota and the levels of SCFAs in women undergoing ART decreased. The application of ART shaped the microbial composition and metabolism, which may provide critical information for understanding the biological changes that occur in women with assisted reproduction.