Fluorosis is a widespread endemic disease. Reports have shown that high fluoride causes the dysfunction of central nervous system (CNS) in animals. The neurotoxicity of fluoride may be related to the activation of microglia. Moreover, numerous studies have found that exercise facilitates the plasticity of structure and function in CNS, partly owing to the regulation of microglia activation. The present study was conducted to explore the effect of exercise on the microglial activation of hippocampus in fluorosis mice. One hundred adult female Institute of Cancer Research (ICR) mice were randomly divided into 4 groups: control group (group C, distilled water by gavage); exercise group (group E, distilled water by gavage and treadmill exercise); fluoride group [group F, 24 mg/kg sodium fluoride (NaF) by gavage]; fluoride plus exercise group (group F + E, 24 mg/kg NaF by gavage and treadmill exercise). After 8 weeks, hippocampal morphological structure, microglial activation and RNA transcriptome of mice in each group were evaluated by hematoxylin and eosin (HE) staining, Nissl staining, immunohistochemistry (IHC), quantitative real time PCR (QRT-PCR) and transcriptome sequencing. We discovered that the number of M1-type microglia in fluorosis-mice hippocampus was significantly increased when compared to group C; group F + E showed a decrease in the number of M1-type microglia with the comparison to group F. In addition, the hippocampal transcriptome analysis showed that 576 differential expression genes (DEG) were confirmed in group F, compared to group C, and 670 DEG were differently expressed in group F + E when compared to group F. Gene Ontology (GO) analysis showed that changed genes were implicated in regulation of transcription, DNA-templated, integral component of membrane and adenosine triphosphate (ATP) binding. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of 670 DEG was helpful to find neuroactive ligand-receptor interaction pathway. In conclusion, these results indicate that treadmill running inhibits the excessive activation of microglia in hippocampus of the fluoride-toxic mice, accompanied with the alteration of neuroactive ligand-receptor interaction pathway.