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Background The real-world exposure levels of non-therapeutic antibiotics and neonicotinoids in type 1 diabetes (T1D) children and their associations as environmental triggers through gut microbiota shifts remained unknown. We thus investigated the antibiotics and neonicotinoids’ exposure levels and their associations with gut microbiota in pediatric T1D. Methods Fifty-one newly onset T1D children along with 67 age-matched healthy controls were recruited. Urine concentrations of 28 antibiotics and 12 neonicotinoids were measured by mass spectrometry. Children were grouped according to the kinds of antibiotics’ and neonicotinoids’ exposures, respectively. The 16S rRNA of fecal gut microbiota was sequenced, and the correlation with urine antibiotics and neonicotinoids’ concentrations was analyzed. Results The overall detection rates of antibiotics were 72.5% and 61.2% among T1D and healthy children, whereas the neonicotinoids detection rates were 70.6% and 52.2% (P = 0.044). Children exposed to one kind of antibiotic or two or more kinds of neonicotinoids had higher risk of T1D, with the odd ratios of 2.579 and 3.911. Furthermore, co-exposure to antibiotics and neonicotinoids was associated with T1D, with the odd ratio of 4.924. Antibiotics or neonicotinoids exposure did not affect overall richness and diversity of gut microbiota. However, children who were exposed to neither antibiotics nor neonicotinoids had higher abundance of Lachnospiraceae than children who were exposed to antibiotics and neonicotinoids alone or together. Conclusion High antibiotics and neonicotinoids exposures were found in T1D children, and they were associated with changes in gut microbiota featured with lower abundance of butyrate-producing genera, which might increase the risk of T1D.