This study addresses the gap in translatable in vitro models for investigating Enterohemorrhagic E. coli (EHEC) infections, particularly relevant to both canine and human health. EHEC is known to induce acute colitis in dogs, leading to symptoms like hemorrhagic diarrhea and hemolytic uremic syndrome, similar to those observed in humans. However, understanding the pathophysiology and developing treatment strategies have been challenging due to the lack of effective models that replicate the clinical disease caused by EHEC in both species. Our approach involved the development of colonoid-derived monolayers using intestinal tissues from healthy, client-owned dogs. These monolayers were exposed to EHEC and the impact of EHEC was assessed through several techniques, including trans-epithelial electrical resistance (TEER) measurement, immunofluorescence staining for junction proteins and mucus, and scanning electron microscopy for morphological analysis. Modified culture with saline, which was intended to prevent bacterial overgrowth, maintained barrier integrity and cell differentiation. EHEC infection led to significant decreases in TEER and ZO-1 expression, but not in E- cadherin levels or mucus production. Additionally, EHEC elicited a notable increase in TNF-α production, highlighting its distinct impact on canine intestinal epithelial cells compared to non-pathogenic E. coli . These findings closely replicate in vivo observations in dogs and humans with EHEC enteropathy, validating the canine colonoid-derived monolayer system as a translational model to study host- pathogen interactions in EHEC and potentially other clinically significant enteric pathogens.