Inflammatory diseases influence tissue metabolism, altering regulation of extracellular adenine nucleotides, with a resultant protective influence of adenosine. Ecto-5′-nucleotidase (CD73) is a central surface enzyme generating extracellular adenosine. Thus, we hypothesized that CD73 is protective in mucosal inflammation as modeled by trinitrobenzene sulfonate (TNBS) colitis. Initial studies revealed a> 3-fold induction of CD73 mRNA levels after TNBS colitis. Additionally, the severity of colitis was increased, as determined by weight loss and colonic shortening, in cd73−/− mice relative to cd73+/+ controls. Likewise, enteral administration of the selective CD73 inhibitor α, β-methylene ADP to cd73+/+ mice resulted in a similar increase in severity of TNBS colitis. Gene array profiling of cytokine mRNA expression, verified by real-time PCR, revealed a> 90% down-regulation of IFN-αA in cd73−/− mice and α, β-methylene ADP-treated cd73+/+ mice, compared with cd73+/+ mice. Exogenous administration of recombinant IFN-αA partially protected TNBS-treated cd73−/− mice. Cytokine profiling revealed similar increases in both IFN-γ and TNF-α mRNA in colitic animals, independent of genotype. However, IL-10 mRNA increased in wild-type mice on day 3 after TNBS administration, whereas cd73−/− mice mounted no IL-10 response. This IL-10 response was restored in the cd73−/− mice by exogenous IFN-αA. Further cytokine profiling revealed that this IL-10 induction is preceded by a transient IFN-αA induction on day 2 after TNBS exposure. Together, these studies indicate a critical regulatory role for CD73-modulated IFNαA in the acute inflammatory phase of TNBS colitis, thereby implicating IFN-αA as a protective element of adenosine signaling during mucosal inflammation.