TRPML3 is an inward rectifying Ca(2+) channel that is regulated by extracytosolic H(+). Although gain-of-function mutation in TRPML3 causes the varitint-waddler phenotype, the role of TRPML3 in cellular physiology is not known. In this study, we report that TRPML3 is a prominent regulator of endocytosis, membrane trafficking and autophagy. Gradient fractionation and confocal localization reveal that TRPML3 is expressed in the plasma membrane and multiple intracellular compartments. However, expression of TRPML3 is dynamic, with accumulation of TRPML3 in the plasma membrane upon inhibition of endocytosis, and recruitment of TRPML3 to autophagosomes upon induction of autophagy. Accordingly, overexpression of TRPML3 leads to reduced constitutive and regulated endocytosis, increased autophagy and marked exacerbation of autophagy evoked by various cell stressors with nearly complete recruitment of TRPML3 into the autophagosomes. Importantly, both knockdown of TRPML3 by siRNA and expression of the channel-dead dominant negative TRPML3(D458K) have a reciprocal effect, reducing endocytosis and autophagy. These findings reveal a prominent role for TRPML3 in regulating endocytosis, membrane trafficking and autophagy, perhaps by controlling the Ca(2+) in the vicinity of cellular organelles that is necessary to regulate these cellular events.