Chemokines induce calcium (Ca 2+) signaling and chemotaxis in dendritic cells (DCs), but the molecular players involved in shaping intracellular Ca 2+ changes remain to be characterized. Using siRNA and knockout mice, we show that in addition to inositol 1, 4, 5-trisphosphate (IP 3)-mediated Ca 2+ release and store-operated Ca 2+ entry (SOCE), the transient receptor potential melastatin 2 (TRPM2) channel contributes to Ca 2+ release but not Ca 2+ influx in mouse DCs. Consistent with these findings, TRPM2 expression in DCs is restricted to endolysosomal vesicles, whereas in neutrophils, the channel localizes to the plasma membrane. TRPM2-deficient DCs show impaired maturation and severely compromised chemokine-activated directional migration as well as bacterial-induced DC trafficking to the draining lymph nodes. Defective DC chemotaxis is due to perturbed chemokine-receptor-initiated Ca 2+ signaling mechanisms, which include suppression of TRPM2-mediated Ca 2+ release and secondary modification of SOCE. DCs deficient in both TRPM2 and IP 3 receptor signaling lose their ability to perform chemotaxis entirely. These results highlight TRPM2 as a key player regulating DC chemotaxis through its function as Ca 2+ release channel and confirm ADP-ribose as a novel second messenger for intracellular Ca 2+ mobilization.—Sumoza-Toledo, A., Lange, I., Cortado, H., Bhagat, H., Mori, Y., Fleig, A., Penner, R., Partida-Sánchez, S. Dendritic cell maturation and chemotaxis is regulated by TRPM2-mediated lysosomal Ca 2+ release.