Unique physiopathological characteristics of tumor tissues impose obstacles to the sufficient penetration of traditional nanomedicines, resulting in undesirable drug delivery efficacy and therapeutic outcomes. Here, we constructed TRAIL-[ND-HCPT]^GAC, a synergistic hydroxycamptothecin (HCPT) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) protein co-loaded disk-shaped nanocarrier with γ-glutamyl transpeptidase responsiveness. When the novel nanodisks extravasated into the tumor interstitium, the γ-glutamyl transpeptidase overexpressed on the tumor cell membranes cleaved the γ-glutamyl portions of the nanodisk surface to produce positively charged amino groups. As a result, the cationic nanodisks possessed stronger tumor infiltration ability through transcytosis than anionic nanodisks. HCPT and TRAIL exerted synergistic antitumor effects with better overall therapeutic efficacy. This TRAIL-[ND-HCPT]^GAC system performed significantly better than free HCPT and remarkably prolonged the survival of breast tumor-bearing mice with no significant toxicity.