Autophagy is an intracellular degradation/recycling process in eukaryotic cells. It contributes to the turnover of cellular components by delivering portions of the cytoplasm and organelles to lysosomes for digestion. The molecular mechanisms of autophagy and vesicle trafficking, especially the biogenesis and turnover of autophagosomes, are poorly understood. In this report, we describe the biological activity of a novel autophagy-related molecule, FLJ30668, or Transmembrane protein 74 (TMEM74). Its transcript was identified by Northern blot and the open reading frame was found to encode 393 amino acids, which shared very little identity with other genetic products. Subcellular localization analysis showed TMEM74 localized to the lysosome and autophagosome. Overexpression of TMEM74 in HeLa cells resulted in autophagic vacuolization, increased the dotted distribution of MDC and GFP-LC3, and endogenous LC3-II levels. Wortmannin, an autophagy inhibitor, partially attenuated these effects. Moreover, knockdown of TMEM74 by small interference RNA abolished the autophagic characteristics induced by starvation. These findings demonstrate that TMEM74 may be involved in promoting functional autophagy during cell starvation and other stress conditions.