Eight G protein-coupled receptors comprise the P2Y receptor family of cell signaling proteins. The goal of the current study was to define native cell signaling pathways regulated by the uridine nucleotide sugar-activated P2Y₁₄ receptor (P2Y₁₄-R). The P2Y₁₄-R was stably expressed in human embryonic kidney (HEK) 293 and C6 rat glioma cells by retroviral infection. Nucleotide sugar-dependent P2Y₁₄-R activation was examined by measuring inhibition of forskolin-stimulated cAMP accumulation. The effect of P2Y₁₄-R activation on mitogen-activated protein kinase signaling also was studied in P2Y₁₄-HEK293 cells and in differentiated HL-60 human myeloid leukemia cells. UDP-Glc, UDP-galactose, UDP-glucuronic acid, and UDP-N-acetylglucosamine promoted inhibition of forskolin-stimulated cAMP accumulation in P2Y₁₄-HEK293 and P2Y₁₄-C6 cells, and this signaling effect was abolished by pretreatment of cells with pertussis toxin. Inhibition of cAMP formation by nucleotide sugars also was observed in direct assays of adenylyl cyclase activity in membranes prepared from P2Y₁₄-C6 cells. UDP-Glc promoted concentration-dependent and pertussis toxin-sensitive extracellular signal-regulated kinase (ERK) 1/2 phosphorylation in P2Y₁₄-HEK293 cells. P2Y₁₄-R mRNA was not observed in wild-type HL-60 cells but was readily detected in dimethyl sulfoxide-differentiated cells. Consistent with this observation, no effect of UDP-Glc was observed in wild-type HL-60 cells, but UDP-Glc-promoted pertussis toxin-sensitive activation of ERK1/2 occurred after differentiation. These results illustrate that the human P2Y₁₄-R signals through G_i to inhibit adenylyl cyclase, and P2Y₁₄-R activation also leads to ERK1/2 activation. This work also identifies two stable P2Y₁₄-R-expressing cell lines and differentiated HL-60 cells as model systems for the study of P2Y₁₄-R-dependent signal transduction.