In the discipline of bottom-up synthetic biology, vesicles define the boundaries of artificial cells and are increasingly being used as biochemical microreactors operating in physiological environments. As the field matures, there is a need to compartmentalize processes in different spatial localities within vesicles, and for these processes to interact with one another. Here we address this by designing and constructing multi-compartment vesicles within which an engineered multi-step enzymatic pathway is carried out. The individual steps are isolated in distinct compartments, and their products traverse into adjacent compartments with the aid of transmembrane protein pores, initiating subsequent steps. Thus, an engineered signalling cascade is recreated in an artificial cellular system. Importantly, by allowing different steps of a chemical pathway to be separated in space, this platform bridges the gap between table-top chemistry and chemistry that is performed within vesicles.