The enteric nervous system consists of more than a million neurons that innervate the GI tract and produce multiple neuroendocrine hormones such as epinephrine, dopamine, and melatonin. Enteric pathogens encounter gradients of these molecules in the GI tract and their virulence is likely to be influenced by these molecules. Previous studies have shown that enterohaemorrhagic E. coli (EHEC) recognize eukaryotic signaling molecules such as the GI tract hormones epinephrine and norepinephrine (catecholamines) to activate the expression of genes involved in infection. Indeed, catecholamines and biogenic amines have been shown to stimulate the growth and adherence of EHEC at discrete concentrations. However, the effect of spatio-temporal gradients of these molecules on colonization and infection have not been studied. The overall goal of this work is to provide fundamental information on how EHEC respond to extracellular signals and gradients of such signals during epithelial cell colonization and infection. We hypothesized that the initial step in EHEC infection (i.e., migration to epithelial cell surfaces) is governed by chemotaxis towards catecholamines such as epinephrine and norepinephrine. Chemotaxis experiments on LB soft agar plates indicated that EHEC migrate towards epinephrine but not norepinephrine. Since different spatio-temporal gradients of eukaryotic molecules are present in the intestinal environment which can impact EHEC attachment and virulence, we also investigated EHEC chemotaxis in response to gradients of different eukaryotic signaling molecules. A microfludic device was developed in which both spatially and temporally stable gradients of signaling molecules can be generated. Our microfludic chemotaxis results demonstrate that eukaroytic signaling molecule gradient determines the extent of EHEC migration and colonization. These results have potential applications in the control of bacterial pathogenesis in infectious diseases and biodefense.