Our previous study showed a reduction of anxiety-like behavior in offspring rats suffered from prenatal cold stress; whether this was related to changes in the offspring gut microbiota is unclear. To obtain the evidence for the role of the gut microbiota in prenatal cold stress offspring, 16S rRNA sequencing technology was used. Male and female offspring rat feces were collected from a room temperature group and a prenatal cold stress group (n ≥ 8) for microbial DNA extraction, followed by 16S rRNA sequencing. The results indicated that prenatal cold stress could change the offspring’s gut microbiota composition. Prenatal cold stress significantly upregulates Lactobacillus, Lactobacillus_gasseri, Bacteroides, and Bacteroides-acidifaciens in female offspring, whereas prenatal cold stress significantly reduced Lachnospiraceae and Prevotellaceae in male offspring. These data showed the characterization of gut microbiota in prenatal cold stress offspring rats, and these data suggest that microbiological intervention in the future can potentially prevent the negative effects caused by cold stress to animals.