Membrane tethering is a fundamental reaction to confer the specificity of membrane trafficking in eukaryotic cells. Although Rab-family GTPases and specific Rab-interacting effector proteins have been reported to be responsible for membrane tethering, whether and how these key components directly and specifically tether subcellular membranes still remain enigmatic. Using the chemically defined systems reconstituted with purified human Rabs and synthetic liposomes, we now establish that Rab-family GTPases have the conserved function to directly trigger membrane tethering, even in the absence of any types of Rab effectors. Furthermore, we strikingly demonstrate that membrane tethering mediated by endosomal Rab11a is selectively stimulated by the cognate Rab effectors, class V myosins, in a GTP-dependent manner. These findings postulate the novel concept that Rab proteins are a bona fide membrane tether to physically link two distinct lipid bilayers, and Rab effectors, including class V myosins, are rather a regulator of Rab-mediated membrane tethering.