Melastatin-related transient receptor potential channel 2 (TRPM2) is a Ca²⁺-permeable, nonselective cation channel that is involved in oxidative stress-induced cell death and inflammation processes. Although TRPM2 can be activated by ADP-ribose (ADPR) in vitro, it was unknown how TRPM2 is gated in vivo. Moreover, several alternative spliced isoforms of TRPM2 identified recently are insensitive to ADPR, and their gating mechanisms remain unclear. Here, we report that intracellular Ca²⁺ ([Ca²⁺]_i) can activate TRPM2 as well as its spliced isoforms. We demonstrate that TRPM2 mutants with disrupted ADPR-binding sites can be activated readily by [Ca²⁺]_i, indicating that [Ca²⁺]_i gating of TRPM2 is independent of ADPR. The mechanism by which [Ca²⁺]_i activates TRPM2 is via a calmodulin (CaM)-binding domain in the N terminus of TRPM2. Whereas Ca²⁺-mediated TRPM2 activation is independent of ADPR and ADPR-binding sites, both [Ca²⁺]_i and the CaM-binding motif are required for ADPR-mediated TRPM2 gating. Importantly, we demonstrate that intracellular Ca²⁺ release activates both recombinant and endogenous TRPM2 in intact cells. Moreover, receptor activation-induced Ca²⁺ release is capable of activating TRPM2. These results indicate that [Ca²⁺]_i is a key activator of TRPM2 and the only known activator of the spliced isoforms of TRPM2. Our findings suggest that [Ca²⁺]_i-mediated activation of TRPM2 and its alternative spliced isoforms may represent a major gating mechanism in vivo, therefore conferring important physiological and pathological functions of TRPM2 and its spliced isoforms in response to elevation of [Ca²⁺]_i.