Two-pore channels (TCPS) are NAADP-sensitive receptor channels that conduct Ca(2+) efflux from the intracellular stores. Discharge of the internal Ca(2+) pools results in the activation of store-operated Ca(2+) entry (SOCE); however, the role of TPCs in the modulation of SOCE remains unexplored. Mammalian cells express three TPCs: TPC1, TPC2 and TPC3, a pseudogene in humans. Here we report that MEG01 and HEK293 cells endogenously express TPC1 and TPC2. Silencing TPC2 expression results in attenuation of the rate and extent of thapsigargin (TG)-evoked SOCE both in MEG01 and HEK293 cells, without having any effect on the ability of cells to accumulate Ca(2+) into the TG-sensitive stores. Similarly, silencing of native TPC2 expression reduced thrombin-induced Ca(2+) entry both in MEG01 and HEK293 cells. Biotinylation analysis revealed that TPC1 and TPC2 are expressed in internal membranes. Finally, co-immunoprecipitation experiments indicated that endogenously expressed TPC2, but not TPC1, assoicates STIM1 and Orai1, but not with TRPC1, in MEG01 cells with depleted intracellular Ca(2+) stores, but not in resting cells. These results provide strong evidence for modulation of SOCE by TPC2 involving de novo association between TPC2 and STIM1, as well as Orai1, in human cells.