Epidemiological investigations reveal that high fluoride and low iodine have strong adverse effects on the intelligence quotient (IQ) of children. Studies also report that in some high fluoride areas, iodine deficiency also exists, especially in China. Here, with the proteomic techniques, we first report on the proteomic changes in brain proteins in offspring rats at postnatal day 20 exposed to high fluoride and/or low iodine. To investigate molecular mechanisms of central neural system injury induced by the above two elements, proteins were isolated and profiled by two-dimensional gel electrophoresis (2DE). By the analysis of Image-Master 2D Elite software, 71 protein spots in 2DE gels of treatment groups were gained and up- or down-regulated by two folds, and 5 proteins were regulated by five folds, with the comparison to the control group. The proteins changed by five folds were identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). The identified proteins are mainly related with cellular signaling, energy metabolism, and protein metabolism and provide a valuable clue to explore the mechanism underlining the neurotoxicity of high fluoride and low iodine. Moreover, these results could provide potential biomarkers for hazards caused by excessive fluoride and low iodine.