Supramolecular binary vesicles based on the host-guest complexation of water-soluble pillar[6]arene (WP6) and SAINT molecule have been successfully constructed, which showed pH-, Ca2+, and thermal-responsiveness. These supramolecular vesicles can efficiently encapsulate model substrate calcein, which then can be efficiently released either by adjusting the solution pH to acidic condition due to the complete disruption of vesicular structure, or particularly, by adding a certain amount of Ca2+ due to the Ca2+-induced vesicle fusion and accompanied by the structure disruption. More importantly, drug loading and releasing experiments demonstrate that an anticancer drug, DOX, can be successfully encapsulated by the supramolecular vesicles, and the resulting DOX-loaded vesicles exhibit efficient release of the encapsulated DOX with the pH adjustment or the introduction of Ca2+. Cytotoxicity experiments suggest that the resulting DOX-loaded supramolecular vesides exhibit comparable therapeutic effect for cancer cells as free DOX and the remarkably reduced damage for normal cells as well. The present multistimuli-responsive supramolecular vesicles have great potential applications in the field of controlled drug delivery. In addition, giant supramolecular vesicles (similar to 3 mu m) with large internal volume and good stability can be achieved by increasing the temperature of WP6 superset of SAINT vesicular solution, and they might have potential applications for bioimaging.