Transient receptor potential (TRP) mucolipins (TRPMLs), encoded by the MCOLN genes, are patho-physiologically relevant endo-lysosomal ion channels crucial for membrane trafficking. Several lines of evidence suggest that TRPMLs mediate localised Ca²⁺ release but their role in Ca²⁺ signalling is not clear. Here, we show that activation of endogenous and recombinant TRPMLs with synthetic agonists evoked global Ca²⁺ signals in human cells. These signals were blocked by a dominant-negative TRPML1 construct and a TRPML antagonist. We further show that, despite a predominant lysosomal localisation, TRPML1 supports both Ca²⁺ release and Ca²⁺ entry. Ca²⁺ release required lysosomal and ER Ca²⁺ stores suggesting that TRPMLs, like other endo-lysosomal Ca²⁺ channels, are capable of ‘chatter’ with ER Ca²⁺ channels. Our data identify new modalities for TRPML1 action.