We assessed the virulence potential and severity of illness associated with non-O157 Shiga toxin-producing E. coli (STEC) strains in the mitomycin C-treated lethal mouse model. STEC were isolated from diverse samples (animal stool, meat and human stool) collected during a large survey in north India. Forty one isolates 10 animal stool, 10 meat and 21 human stool were selected based on profiling of virulence genes and tested for colonization potential, Shiga toxin concentration in the intestinal contents, hematological and renal function parameters and tissue histopathology. Though cattle stool isolates were better colonizers, there was significantly less pathology in animals infected with them as compared to human and meat isolates. Meat and human isolates could produce pathology at lower levels of colonization. Thrombocytopenia and an increase in blood urea nitrogen were significantly associated with pathology (P<0.05), whereas Hb and creatinine levels were not (p>0.05). Stx2 production was significantly correlated with thrombocytopenia, and both Stx1 and Stx2 were significantly correlated with an increase in BUN. Pathology was observed in 2, 5 and 13 cattle stool, meat and human stool isolates, respectively. Some isolates were associated with more severe pathologies, i.e. thrombotic microangiopathy (TMA), tubular and smooth muscle vacuolation, acute tubular necrosis and neuronal ischaemia. Severe pathologies were more significantly associated with isolates that produced both Stx1 and Stx2 than with isolates that produced either alone. We also conclude that the cattle and meat isolates have the potential to cause severe illness, thereby posing a significant public health threat.