Inosine is an endogenous purine nucleoside that is produced by catabolism of adenosine. Adenosine has a short half-life (approximately 10 s) and is rapidly deaminated to inosine, a stable metabolite with a half-life of approximately 15 h. Resembling adenosine, inosine acting through adenosine receptors (ARs) exerts a wide range of anti-inflammatory and immunomodulatory effects in vivo. The immunomodulatory effects of inosine in vivo, at least in part, are mediated via the adenosine A_2A receptor (A_2AR), an observation that cannot be explained fully by in vitro pharmacological characterization of inosine at the A_2AR. It is unclear whether the in vivo effects of inosine are due to inosine or a metabolite of inosine engaging the A_2AR. Here, utilizing a combination of label-free, cell-based, and membrane-based functional assays in conjunction with an equilibrium agonist-binding assay we provide evidence for inosine engagement at the A_2AR and subsequent activation of downstream signaling events. Inosine-mediated A_2AR activation leads to cAMP production with an EC₅₀ of 300.7 μM and to extracellular signal-regulated kinase-1 and -2 (ERK1/2) phosphorylation with an EC₅₀ of 89.38 μM. Our data demonstrate that inosine produces ERK1/2-biased signaling whereas adenosine produces cAMP-biased signaling at the A_2AR, highlighting pharmacological differences between these two agonists. Given the in vivo stability of inosine, our data suggest an additional, previously unrecognized, mechanism that utilizes inosine to functionally amplify and prolong A_2AR activation in vivo.