Neonicotinoids exposure was found to induce thyroid dysfunction. However, there lack of direct evidence between neonicotinoids exposure and thyroid hormone (TH) disruption in population study, especially in children, which limits the understanding on their health hazard. To fill this knowledge gap, we conducted a cross-sectional study on children of a rural area in South China (n = 88), and analyzed urinary ten neonicotinoids (including metabolites), serum TH, thyroxine-binding globulin (TBG), and thyroid stimulating hormone (TSH) levels. Based on linear regression, generalized additive model, and Bayesian kernel machine regression, neonicotinoids levels were found to be correlated with TH, TBG, and TSH levels, with stronger effects for metabolites than parent compounds in most cases. Mixture exposure of neonicotinoids had significantly positive effect on free triiodothyronine (T