Rab GTPases regulate diverse aspects of the biogenesis of secretory and endocytic organelles. The conformational changes that accompany GTP binding and hydrolysis by Rab are harnessed to recruit and activate specific effector proteins that regulate vesicle tethering and fusion, cargo sorting, and cytoskeleton-dependent organelle transport. We review recent progress in understanding how Rab GTPases are regulated and how they regulate membrane trafficking.