A tumour microenvironment imposes barriers to the passive diffusion of molecules, which renders tumour penetration an unresolved obstacle to an effective anticancer drug delivery. Here, we present a gamma-glutamyl transpeptidase-responsive camptothecin-polymer conjugate that actively infiltrates throughout the tumour tissue through transcytosis. When the conjugate passes on the luminal endothelial cells of the tumour blood vessels or extravasates into the tumour interstitium, the overexpressed gamma-glutamyl transpeptidase on the cell membrane cleaves the gamma-glutamyl moieties of the conjugate to generate positively charged primary amines. The resulting cationic conjugate undergoes caveolae-mediated endocytosis and transcytosis, which enables transendothelial and transcellular transport and a relatively uniform distribution throughout the tumour. The conjugate showed a potent antitumour activity in mouse models that led to the eradication of small solid tumours (similar to 100 mm(3)) and regression of large established tumours with clinically relevant sizes (similar to 500 mm(3)), and significantly extended the survival of orthotopic pancreatic tumour-bearing mice compared to that with the first-line chemotherapeutic drug gemcitabine.