BACKGROUND: Microplastics (MPs) have garnered widespread attention because of their presence in human placenta, stool, and even blood. Ingestion is considered the major route of human exposure to MPs. It has been found that the consumption of food and water is associated with more MP abundance in human stools. The usage of plastic containers, particularly feeding bottles, may be a major contributor to MP contamination. However, human exposure to MPs and potential factors that influence exposure, especially for preschoolers, remains largely unknown. When exposed to MPs, mice exhibited gut microbiota dysbiosis, including alterations in diversity indices, a decreased relative abundance of probiotics and an increased abundance of pathogenic bacteria. Such results have also been observed in human gut in vitro models, however, the actual association between MP exposure and human intestinal microbiota remains unclear. Therefore, this study aimed to evaluate MP concentrations in preschoolers' stools, explore possible dietary factors that influence preschooler exposure to MPs, and investigate their potential association with the gut microbiota. METHODS: A cross-sectional study was conducted in Xiamen, China in October 2022. We investigated the feeding behaviours and dietary habits of preschool children. A total of 69 couples of stool samples were collected and analyzed for MPs test and gut microbiota analysis. Pyrolysis-gas chromatography coupled with mass spectrometry (Py-GC/MS) was used for quantifying 11 types of MPs. The gut microbiota composition was analyzed by 16S rRNA gene sequencing. FINDINGS: The results showed that only polyvinyl chloride (PVC), polyethylene terephthalate (PET), polyethylene (PE), and polyamide 6 (PA6) were detected in 85.5% stool samples, with concentrations of 317.4 (152.0, 491.9) μg/g dw, 299.0 (196.1, 619.9) μg/g dw, 206.2 (154.1, 240.3) μg/g dw, and 17.9 (13.4, 18.6) μg/g dw, respectively. The median estimated daily intake (EDI) for preschoolers was 425.9 (272.5, 762.3) μg/kg-bw/d. Dairy intake may influence MP concentration in preschoolers' stools, and the usage of feeding bottles may be a specific source of MP contamination. Moreover, higher PVC concentrations were observed in the stools when the children took more time to eat a meal. MP exposure was inversely associated with alpha indices and possibly affected certain probiotic taxa, such as Parabacteroides and Alistipes, in preschool children. INTERPRETATION: Our data provided baseline evidence for MP exposure doses and potential dietary factors that may influence MP exposure in preschoolers. These findings supported the perspective that MP exposure might be associated with the disturbance of gut microbiota. Further studies focusing on sensitive populations with larger sample sizes are needed. FUNDING: This study was funded by the National Natural Science Foundation of China (grant number: 82003412), the Shanghai Municipal Health Commission (grant number: 20214Y0019), and the Project of Shanghai Municipal Financial Professional foundation (Food Safety Risk Assessment) (grant number: RA-2022-06).