Interleukin 3 (IL-3) plays an important role in hematopoiesis and immune regulation, brain IL-3/IL-3R signaling has been shown to involve in the physiological and pathological processes of a variety of neurodegenerative diseases, but its role in prion diseases is rarely described. Here, the changes of IL-3/IL-3R and its downstream signaling pathways in a scrapie-infected cell line and in the brains of several scrapie-infected rodent models were evaluated by various methods. Markedly decreased IL-3R & alpha; were observed in the brains of scrapie-infected rodents at terminal stage and in the prion-infected cell model, which showed increased in the brain samples collected at early and middle stage of infection. The IL-3 levels were almost unchanged in the brains of scrapie-infected mice and in the prion-infected cell line. Morphological assays identified close co-localization of the increased IL-3R & alpha; signals with NeuN- and Iba1-positive cells, whereas co-localization of IL-3 signals with NeuN- and GFAP-positive cells in the scrapie-infected brain tissues. Some downstream components of IL-3/IL-3R pathways, including JAK2-STAT5 and PI3K/AKT/mTOR pathways, were downregulated in the brains of scrapie-infected rodents at terminal stage and in the prion-infected cells. Stimulation of recombinant IL-3 on the cultured cells showed prion that the prion-infected cells displayed markedly more reluctant responses of JAK2-STAT5 and PI3K/AKT/mTOR pathways than the normal partner cells. These data suggest that although prion infection or PrPSc accumulation in brain tissues does not affect IL-3 expression, it significantly downregulates IL-3R levels, thereby inhibiting the downstream pathways of IL-3/IL-3R and blocking the neuroregulatory and neuroprotective activities of IL-3.