We recently showed that A_2A adenosine receptor activation by endogenous adenosine contributes to interleukin-10 (IL-10) production in polymicrobial sepsis. Here we investigated the molecular mechanisms underpinning this interaction between adenosine receptor signaling and infection by exposing macrophages to Escherichia coli. We demonstrated using receptor knockout mice that A_2A receptor activation is critically required for the stimulatory effect of adenosine on IL-10 production by E coli–challenged macrophages, whereas A_2B receptors have a minor role. The stimulatory effect of adenosine on E coli–induced IL-10 production did not require toll-like receptor 4 (TLR4) or MyD88, but was blocked by p38 inhibition. Using shRNA we demonstrated that TRAF6 impairs the potentiating effect of adenosine. Measuring IL-10 mRNA abundance and transfection with an IL-10 promoter-luciferase construct indicated that E coli and adenosine synergistically activate IL-10 transcription. Sequential deletion analysis and site-directed mutagenesis of the IL-10 promoter revealed that a region harboring C/EBP binding elements was responsible for the stimulatory effect of adenosine on E coli–induced IL-10 promoter activity. Adenosine augmented E coli–induced nuclear accumulation and DNA binding of C/EBPβ. C/EBPβ-deficient macrophages failed to produce IL-10 in response to adenosine and E coli. Our results suggest that the A_2A receptor–C/EBPβ axis is critical for IL-10 production after bacterial infection.