TRPM2 is a Ca²⁺-permeable channel activated by oxidative stress or TNF-α, and TRPM2 activation confers susceptibility to cell death. The mechanisms were examined here in human monocytic U937-ecoR cells. This cell line expresses full-length TRPM2 (TRPM2-L) and several isoforms including a short splice variant lacking the Ca²⁺-permeable pore region (TRPM2-S), which functions as a dominant negative. Treatment with H₂O₂, a model of oxidative stress, or TNF-α results in reduced cell viability. Expression of TRPM2-L and TRPM2-S was modulated by retroviral infection. U937-ecoR cells expressing increased levels of TRPM2-L were treated with H₂O₂ or TNF-α, and these cells exhibited significantly increased intracellular calcium concentration ([Ca²⁺]_i), decreased viability, and increased apoptosis. A dramatic increase in cleavage of caspases-8, -9, -3, and -7 and poly(ADP-ribose)polymerase (PARP) was observed, demonstrating a downstream mechanism through which cell death is mediated. Bcl-2 levels were unchanged. Inhibition of the [Ca²⁺]_i rise with the intracellular Ca²⁺ chelator BAPTA blocked caspase/PARP cleavage and cell death induced after activation of TRPM2-L, demonstrating the critical role of [Ca²⁺]_i in mediating these effects. Downregulation of endogenous TRPM2 by RNA interference or increased expression of TRPM2-S inhibited the rise in [Ca²⁺]_i, enhanced cell viability, and reduced numbers of apoptotic cells after exposure to oxidative stress or TNF-α, demonstrating the physiological importance of TRPM2. Our data show that one mechanism through which oxidative stress or TNF-α mediates cell death is activation of TRPM2, resulting in increased [Ca²⁺]_i, followed by caspase activation and PARP cleavage. Inhibition of TRPM2-L function by reduction in TRPM2 levels, interaction with TRPM2-S, or Ca²⁺ chelation antagonizes this important cell death pathway.