To better understand the role of host cell‐derived molecules on enterohemorrhagic Escherichia coli (EHEC) infection, we studied EHEC virulence gene expression when exposed to cell‐free spent (conditioned) medium (CM) from HCT‐8 intestinal epithelial cells. Exposure to HCT‐8 CM for 1 h and 3 h increased the expression of 32 of 41 EHEC locus of enterocyte effacement (LEE) virulence genes compared with fresh medium (FM). Expression of the Shiga toxin 1 (stx1B) gene was up‐regulated at 1 h of exposure. Seventeen genes encoded by prophage 933W, including those for Stx2, were also up‐regulated at both time‐points. The increase in 933W prophage expression was mirrored by a 2.7‐fold increase in phage titers. Consistent with the increase in virulence gene expression, we observed a fivefold increase in EHEC attachment to epithelial cells when exposed to CM. The increase in EHEC attachment was abolished when CM was heated to 95 °C or treated with proteinase K to degrade the proteins. The host cell‐derived molecule(s) were larger than 3 kDa, which suggests that the molecule(s) that increase EHEC virulence and attachment are protein‐based.