Copper (Cu2+) is vital for proper brain function, serving as a cofactor in essential biological processes. However, excessive levels of Cu2+ can disrupt redox homeostasis, resulting in oxidative stress. TRPM2 channel is a Ca2+-permeable channel that is highly sensitive to oxidative stress and is known to be critical in mediating cellular processes. The signaling mechanisms involved in TRPM2 channel activation, which lead to microglial cell activation, remain poorly elucidated. Therefore, in the present study, we investigated the mechanisms underlying TRPM2 channel and microglial activation induced by Cu2+. Treatment of Cu2+ for 8 h significantly increased microglial intracellular Ca2+ concentration ([Ca2+]i), and such results were prevented by the presence of TRPM2 inhibitors, 2-APB and ACA. Extended exposure to Cu2+ (10-100 μM) for 24 h induced microglial cell activation, as exemplified by the morphological changes from elongated and small cell body to amoeboid-like shape. This Cu2+-induced microglial activation was remarkably inhibited by TRPM2 blockers. Poly(ADP-ribose) polymerase 1 (PARP-1) activates the TRPM2 channel by producing its primary activator, ADP-ribose (ADPR). Here, we found that the increases in [Ca2+]i and microglial morphological changes were attenuated by PJ-34 and DPQ, PARP-1 inhibitors. In addition, a noticeable increase in intracellular ROS level was observed in microglial cells induced by Cu2+, and such an increase was suppressed by the inhibition of protein kinase C (PKC) and NADPH oxidase (NOX). Interestingly, the increases in [Ca2+]i and microglial activation were also attenuated by the PKC and NOX inhibitors. Altogether, these data suggest that PKC and NOX mediate ROS production, leading to the PARP-dependent TRPM2 activation, which subsequently results in a Ca2+ response that is responsible for microglial cell activation.