The development of highly efficient tumor-targeted delivery systems is crucial for successful tumor treatment. Previously, a novel cell-penetrating peptide TAT and cleavable polyethylene glycol (PEG) co-modified liposome delivery system (C-TAT-Lipo) showed enhanced accumulation in tumor regions. Under the control of cysteine (Cys), the liposomes were activated extracellularly and achieved increased delivery of their cargo into tumor cells efficiently. In this study, we developed an optimal formulation for the encapsulation of Doxorubicin (DOX) by this delivery system for tumor treatment. The in vitro study showed that the C-TAT-Lipo with Cys delivery system not only enhanced the amount of DOX delivered by at least 100% compared to other DOX-containing formulations, but also displayed high cytotoxicity against tumorigenic cell lines. Compared to other groups, the DOX-loaded C-TAT-Lipo formulation in the presence of cysteine enhanced treatment efficacy by lowering the IC50 (1.67 +/- 0.14 microM) and increasing the cancer cell apoptosis percentage (37.10%). Moreover, the in vivo antitumor activity also showed that DOX-loaded C-TAT-Lipo with injection of cysteine achieved the best tumor growth inhibition with a tumor growth rate of only 58.40 +/- 16.33% (% of initial volume/day), which was significant less than that achieved by other DOX formulations.