Aberrant ligand-independent G protein–coupled receptor constitutive activity has been implicated in the pathophysiology of a number of cancers. The adenosine A_2B receptor (A_2BAR) is dynamically upregulated under pathologic conditions associated with a hypoxic microenvironment, including solid tumors. This, in turn, may amplify ligand-independent A_2BAR signal transduction. The contribution of A_2BAR constitutive activity to disease progression is currently unknown yet of fundamental importance, as the preferred therapeutic modality for drugs designed to reduce A_2BAR constitutive activity would be inverse agonism as opposed to neutral antagonism. The current study investigated A_2BAR constitutive activity in a heterologous expression system and a native 22Rv1 human prostate cancer cell line exposed to hypoxic conditions (2% O₂). The A_2BAR inverse agonists, ZM241385 [4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol] or PSB-603 (8-(4-(4-(4-chlorophenyl)piperazide-1-sulfonyl)phenyl)-1-propylxanthine), mediated a concentration-dependent decrease in baseline cAMP levels in both cellular systems. Proliferation of multiple prostate cancer cell lines was also attenuated in the presence of PSB-603. Importantly, both the decrease in baseline cAMP accumulation and the reduction of proliferation were not influenced by the addition of adenosine deaminase, demonstrating that these effects are not dependent on stimulation of A_2BARs by the endogenous agonist adenosine. Our study is the first to reveal that wild-type human A_2BARs have high constitutive activity in both model and native cells. Furthermore, our findings demonstrate that this ligand-independent A_2BAR constitutive activity is sufficient to promote prostate cancer cell proliferation in vitro. More broadly, A_2BAR constitutive activity may have wider, currently unappreciated implications in pathologic conditions associated with a hypoxic microenvironment.