Synthetic cells， artificial cell-like particles， capable of autonomously synthesizing RNA and proteins based on a DNA template， are emerging platforms for studying cellular functions and for revealing the origins-of-life. Here， it is shown for the first time that artificial lipid-based vesicles， containing the molecular machinery necessary for transcription and translation， can be used to synthesize anticancer proteins inside tumors. The synthetic cells are engineered as stand-alone systems， sourcing nutrients from their biological microenvironment to trigger protein synthesis. When pre-loaded with template DNA， amino acids and energy-supplying molecules， up to 2 × 10 copies of green fluorescent protein are synthesized in each synthetic cell. A variety of proteins， having molecular weights reaching 66 kDa and with diagnostic and therapeutic activities， are synthesized inside the particles. Incubating synthetic cells， encoded to secrete Pseudomonas exotoxin A (PE) with 4T1 breast cancer cells in culture， resulted in killing of most of the malignant cells. In mice bearing 4T1 tumors， histological evaluation of the tumor tissue after a local injection of PE-producing particles indicates robust apoptosis. Synthetic cells are new platforms for synthesizing therapeutic proteins on-demand in diseased tissues.