ADP is the endogenous agonist for both P2Y₁ and P2Y₁₂ receptors, which are important therapeutic targets. It was previously demonstrated that ADP and a synthetic agonist, 2-methylthioadenosine 5′-diphosphate (2MeSADP), can induce apoptosis by activating the human P2Y₁ receptor heterologously expressed in astrocytoma cells. However, it was not known whether the P2Y₁₂ receptor behaved similarly. We demonstrated here that, unlike with the G_q-coupled P2Y₁ receptor, activation of the G_i-coupled P2Y₁₂ receptor does not induce apoptosis. Furthermore, activation of the P2Y₁₂ receptor by either ADP or 2MeSADP significantly attenuates the tumor necrosis factor α (TNFα)-induced apoptosis in 1321N1 human astrocytoma cells. This protective effect was blocked by the P2Y₁₂ receptor antagonist 2-methylthioAMP and by inhibitors of phospholipase C (U73122) and protein kinase C (chelerythrin), but not by the P2Y₁ receptor antagonist MRS2179. Toward a greater mechanistic understanding, we showed that hP2Y₁₂ receptor activation by 10nM 2MeSADP, activates Erk1/2, Akt, and JNK by phosphorylation. However, at a lower protective concentration of 100pM 2MeSADP, activation of the hP2Y₁₂ receptor involves only phosphorylated Erk1/2, but not Akt or JNK. This activation is hypothesized as the major mechanism for the protective effect induced by P2Y₁₂ receptor activation. Apyrase did not affect the ability of TNFα to induce apoptosis in hP2Y₁₂-1321N1 cells, suggesting that the endogenous nucleotides are not involved. These results may have important implications for understanding the signaling cascades that follow activation of P2Y₁ and P2Y₁₂ receptors and their opposing effects on cell death pathways.