Previous research has found a correlation between heavy metals and gut microbiota in humans. However, there are few population-based studies examining the impact of early life thallium (Tl) exposure on neonatal microbiome. 342 newborns were recruited from Hunan Children's Hospital and subsequently divided into three groups (low, medium, and high) based on the 25th and 75th percentiles of serum Tl concentration. Additionally, the relationship between Tl and gut microbiota was analyzed in subgroups (preterm or full-term neonates). The association between Tl and gut microbiota in neonates was analyzed by Redundancy analysis, Spearman correlation analysis and MaAsLin2. The detection rate of Tl in neonates was 100%, with the median concentration of 0.021 μg/L. In all neonates, we found significant differences in the Chao1 and ACE indices of α-diversity in gut microbiota, and the relative abundances of Bacteroidota and Bacteroidetes were significantly different among groups (p <
0.05). Following the covariate adjustment, Tl was negatively correlated with Gemmatimonadota (Coef = 0.265, p <
0.05) in preterm neonates. In full-term neonates, Tl exhibited a positive correlation with the relative abundance of Robinsoniella (Coef = 0.563, p = 0.009) and a negative correlation with that of Pseudomonas (Coef = - 0.592, p = 0.012). Tryptophan and renin-angiotensin system pathways might exert important roles in Tl exposure. This study indicated that Tl exposure was associated with changes in α-diversity and the composition of gut microbiota in neonates, with Gemmatimonadota being predominantly affected in preterm neonates and Robinsoniella and Pseudomonas in full-term neonates.