Inosine is an endogenous nucleoside that is produced by metabolic deamination of adenosine. Inosine is metabolically more stable (half-life 15h) than adenosine (half-life <10s). Inosine exerts anti-inflammatory and immunomodulatory effects similar to those observed with adenosine. These effects are mediated in part through the adenosine A receptor (AR). Relative to adenosine inosine exhibits a lower affinity towards the AR. Therefore, it is generally believed that inosine is incapable of activating the AR through direct engagement, but indirectly activates the AR upon metabolic conversion to higher affinity adenosine. A handful of studies, however, have provided evidence for direct inosine engagement at the AR leading to activation of downstream signaling events and inhibition of cytokine production. Here, we demonstrate that under conditions devoid of adenosine, inosine as well as an analog of inosine 6-S-[(4-Nitrophenyl)methyl]-6-thioinosine selectively and dose-dependently activated AR-mediated cAMP production and ERK1/2 phosphorylation in CHO cells stably expressing the human AR. Inosine also inhibited LPS-stimulated TNF-α, CCL3 and CCL4 production by splenic monocytes in an AR-dependent manner. In addition, we demonstrate that a positive allosteric modulator (PAM) of the AR enhanced inosine-mediated cAMP production, ERK1/2 phosphorylation and inhibition of pro-inflammatory cytokine and chemokine production. The cumulative effects of allosteric enhancement of adenosine-mediated and inosine-mediated AR activation may be the basis for the sustained anti-inflammatory and immunomodulatory effects observed in vivo and thereby provide insights into potential therapeutic interventions for inflammation- and immune-mediated diseases.