Prostate cancer is the most commonly diagnosed and second most lethal malignancy in men, due mainly to a lack of effective treatment for the metastatic disease. A number of recent studies have shown that activation of the purine nucleoside receptor, adenosine A(3) receptor (A(3)AR), attenuates proliferation of melanoma, colon, and prostate cancer cells. In the present study, we determined whether activation of the A(3)AR reduces the ability of prostate cancer cells to migrate in vitro and metastasize in vivo. Using severe combined immunodeficient mice, we show that proliferation and metastasis of AT6.1 rat prostate cancer cells were decreased by the administration of A(3)AR agonist N(6)-(3-iodobenzyl) adenosine-5'-N-methyluronamide. In vitro studies show that activation of A(3)AR decreased high basal nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity present in these cells, along with the expression of Rac1 and p47(phox) subunits of this enzyme. Inhibition of NADPH oxidase activity by the dominant-negative RacN17 or short interfering (si)RNA against p47(phox) reduced both the generation of reactive oxygen species and the invasion of these cells on Matrigel. In addition, we show that membrane association of p47(phox) and activation of NADPH oxidase is dependent on the activity of the extracellular signal-regulated kinase (ERK)1/2 mitogen-activated protein kinase pathway. We also provide evidence that A(3)AR inhibits ERK1/2 activity in prostate cancer cells through inhibition of adenylyl cyclase and protein kinase A. We conclude that activation of the A(3)AR in prostate cancer cells reduces protein kinase A-mediated stimulation of ERK1/2, leading to reduced NADPH oxidase activity and cancer cell invasiveness.