Neutrophils are the most abundant immune cells in the body and provide pivotal defense mechanisms. The role of ion fluxes upon neutrophil activation is increasingly appreciated. TRPM2 is a nonselective cation channel highly expressed in neutrophils and gated by products of oxidative stress, which are predominantly produced by NADPH oxidase (NOX2). Despite this direct link to neutrophil function, TRPM2 activation and its beneficial or detrimental consequences still need to be elucidated. Using murine (C57BL/6) WT and TRPM2-/- bone marrow-derived neutrophils, 3D chemotaxis in gradients of fMLP, KC, LTB4, and C5a was analyzed (N≥3). Absolute intracellular Na+ concentration [Na+]i was measured using sodium-sensitive Asante Natrium Green-2 (ANG-2). [Na+]i upon fMLP stimulation and its dependence on Na+/K+-ATPase and Na+/Ca2+ exchanger (NCX) activity was analyzed (N=3 for each condition). Reactive oxygen species (ROS) production was measured in differentiated siTRPM2-HL-60 cells using dihydrorhodamine (DHR) oxidation assay (N=3). Chemotaxis of TRPM2-/- neutrophils in 3D matrix is not significantly altered when compared to WT neutrophils. After fMLP stimulation [Na+]i rises in neutrophils of both genotypes. It reaches higher levels in TRPM2-/- neutrophils (13.1 ± 0.6 mmol/l vs 15.6 ± 0.9 mmol/l). Addition of ouabain leads to a further increase in [Na+]i. Addition of the NCX inhibitor KB-R7943 lowers [Na+]i in WT neutrophils, but not in TRPM2-/- (16.5 ± 1.2 mmol/l vs 25.4 ± 1.7 mmol/l). Permeabilizing membranes with a Na+ ionophore in the absence of [Na+]o reveals intracellular sodium release, which is higher after fMLP stimulation in TRPM2-/- than in WT neutrophils. TRPM2 knock-down in HL-60 cells leads to diminished ROS production. Values are means ± S.E.M. Although chemotaxis is not altered in TRPM2 knock-out neutrophils, the ionic homeostasis in TRPM2-/- is disrupted. High [Na+]i in activated TRPM2-/- neutrophils hinders NCX transport. NCX activation normally follows neutrophil stimulation and ROS production. Both NCX and NOX2 are electrogenic and thereby modulate ROS production. Our findings suggest that TRPM2 gating upon neutrophil stimulation regulates not only ionic concentrations but the function of NCX and NOX2 and hence downstream effects depending on these proteins.