Objective To analyze the biological characteristics of intestinal flora in hypothyroidism patients with abnormal thyroperoxidase antibodies (TPOAb) in the third trimester of pregnancy and their correlations with maternal and infant outcomes. Methods A total of 40 hypothyroidism patients in the third trimester of pregnancy (20 with abnormal TPOAb level [>35 IU/mL] and 20 with normal TPOAb level [≤35 IU/mL]) who visited in the Department of Obstetrics of our hospital from Sep. 2018 to Oct. 2019 were selected. The species and relative abundance of intestinal flora between the 2 groups were compared by 16S rRNA sequencing, and Bifidobacterium and Lachnospira were quantitatively analyzed by quantitative real-time ploymerase chain reaction (qRT-PCR). In addition, the serological test indexes and maternal and infant clinical indexes of the 2 groups were compared, and the correlation between maternal intestinal flora and maternal and infant outcomes was analyzed. Results 16S rRNA sequencing showed that the relative abundance of Bifidobacterium in the intestinal flora of TPOAb positive pregnant women was increased, while the relative abundance of Lachnospira was decreased. qRT-PCR analysis also confirmed this change. The results of serological indexes showed that the levels of C reactive protein (CRP), interleukin (IL)-2 and IL-6 before delivery and the level of CRP after delivery were significantly higher in the TPOAb positive group than in the TPOAb negative group (all P<0.05). Correlation analysis showed that the relative abundance of Bifidobacterium was positively correlated with the serum TPOAb level (r=0.55, P<0.01), and the relative abundance of Lachnospira was negatively correlated with the serum IL-6 level (r=－0.37, P=0.02). The times of previous embryo suspension and spontaneous abortion of the TPOAb positive group were significantly higher than those of the TPOAb negative group (P=0.04 and 0.02). The neonatal weight and head circumference of the TPOAb positive group were significantly lower than those of the TPOAb negative group (both P<0.01), and the incidences of neonatal asphyxia and neonatal distress during delivery were significantly higher than those of the TPOAb negative group (both P=0.04). Conclusion The abnormal TPOAb of hypothyroid pregnant women in the third trimester of pregnancy is closely related to the intestinal flora and poor maternal and infant outcomes. Intestinal Bifidobacterium and Lachnospira may be noninvasive biomarkers for abnormal TPOAb in pregnant women with hypothyroidism in the third trimester of pregnancy.