G protein-coupled P2Y receptors (P2Y-R) are activated by adenine and uracil nucleotides. The P2Y₁₄ receptor (P2Y₁₄-R) is activated by at least four naturally occurring UDP sugars, with UDP-glucose (UDP-Glc) being the most potent agonist. With the goal of identifying a competitive antagonist for the P2Y₁₄-R, UDP was examined for antagonist activity in COS-7 cells transiently expressing the human P2Y₁₄-R and a chimeric Gα protein that couples Gi-coupled receptors to stimulation of phosphoinositide hydrolysis. UDP antagonized the agonist action of UDP-Glc, and Schild analysis confirmed that the antagonism was competitive (pK_B = 7.28). Uridine 5′-O-thiodiphosphate also antagonized the human P2Y₁₄-R (hP2Y₁₄-R) with an apparent affinity similar to that of UDP. In contrast, no antagonist activity was observed with ADP, CDP, or GDP, and other uracil analogs also failed to exhibit antagonist activity. The antagonist activity of UDP was not observed at other hP2Y-R. In contrast to its antagonist action at the hP2Y₁₄-R, UDP was a potent agonist (EC₅₀ = 0.35 μM) at the rat P2Y₁₄-R. These results identify the first competitive antagonist of the P2Y₁₄-R and demonstrate pharmacological differences between receptor orthologs.