We have previously shown that human intestinal epithelial cell monolayers (Caco-2) subjected to hypoxia and re-oxygenation release soluble factors into the apical media that activate the virulence of the opportunistic pathogen Pseudomonas aeruginosa to express the potent barrier dysregulating protein, the PA-I lectin/adhesin. Here we defined the role of HIF-1 in this response. We tested the ability of media from Caco-2 cells with forced expression of HIF-1 to increase PA-I expression in P. aeruginosa, and found that media from Caco-2 cells overexpressing HIF-1 increased PA-I expression compared to media from control cells (P<0.001 ANOVA). To identify the components responsible for this response, media was fractionated by molecular weight and subjected to mass spectroscopy which identified adenosine as the possible mediator. Both adenosine, and its immediate downstream metabolite inosine, induced PA-I expression in P. aeruginosa in a dose-dependent fashion. Because inosine was not detectable in the media of Caco-2 cells exposed to hypoxia or overexpressing HIF-1, we hypothesized that P. aeruginosa itself might metabolize adenosine to inosine. Using mutant and parental strains of P. aeruginosa, we demonstrated that P. aeruginosa metabolized adenosine to inosine via adenosine deaminase and that the conditioned media enhanced the extracellular accumulation of inosine. Taken together these results provide evidence that P. aeruginosa can recognize and respond to extracellular end-products of intestinal hypoxia that are released following activation of HIF-1. The ability of P. aeruginosa to metabolize adenosine to inosine may represent a subversive microbial virulence strategy that deprives the epithelium of the cytoprotective actions of adenosine.