Mounting evidence suggests a role for the microbiome-gut-brain axis in amyloid-associated neurodegeneration, but the pathogenic changes induced by amyloids in the gastro-intestinal tract remain elusive. To scrutinize the early response to amyloids of human and bacterial origin, we challenged primary murine myenteric networks with Aβ1-42 (vs a scrambled version of Aβ1-42) and curli (vs culture medium), respectively, and performed shotgun RNA sequencing. Both amyloid types induced a transcriptional signature of DNA damage and cell cycle dysregulation. Using in vitro neurosphere-derived cultures and in vivo amyloid injections we found that enteric glia and smooth muscle cells were the most responsive cell types, showing increased proliferation, γH2AX burden and SOD2 levels after amyloid challenge. Consistent with this activated state, we identified a pro-inflammatory hub in the transcriptional profile of amyloid-stimulated myenteric networks. Enteric glia were the principal source of the associated cytokines, and in vivo, this was accompanied by an influx of immune cells. Together, these results shed new light on the intrinsic vulnerability of ENS cells to both amyloid species and position enteric glial cell activation as an early driver of neurodegenerative disease progression.