Microglial cell is a highly plastic cell in which it retracts its branched processes upon activation by structurally diverse molecules. Elevation of these molecules in the brain has been implicated in a diversity of diseases conditions in the CNS, where these molecules promote production of toxicity mediators, such as ROS. Microglial cell activation in response to ROS has been of particular interest. Emerging evidence supports a role for the TRPM2 channel in ROS-induced neuroinflammation. Thus, the current study aims to examine the role of the TRPM2 channel in mediating H2O2-induced microglial activation. A multidisciplinary approach was adopted, including primary microglial isolation, single cell calcium imaging, immunocytochemistry, confocal microscopy and computer-aided analysis of cell morphology. H2O2-induced microglial activation were observed in WT microglial cells but were ablated by genetic or pharmacological inhibition of the TRPM2 channel. Exposure to H2O2 raised the [Ca2+]i via promoting Ca2+ influx, which was prevented by TRPM2-KO. H2O2 induced ROS production and PARP-1 activation.  H2O2induced ROS production and PARP-1 activation as well as an increase in the [Ca2+]i and microglial activation, were suppressed by inhibiting PKC and NOX. Furthermore, H2O2-induced PARP-1 activation, increase in the [Ca2+]i and microglial activation were attenuated by inhibiting the Ca2+-sensitive PYK2 and downstream MEK/ERK kinases. The findings provide strong evidence to support that the TRPM2 channel is functionally expressed and plays a major role in ROS-induced Ca2+ signalling as well as cell activation in microglia. Such information is useful for a better understanding of microglial cells in oxidative stress-related pathologies.