Targeted delivery of anticancer drugs to tumors has attracted considerable research interest because of its potential to reduce adverse toxicity while improving therapeutic efficacy. In this study, we synthesized and evaluated the therapeutic efficacy of a conjugate of a high-affinity peptide (aptide) and the anticancer drug docetaxel (DTX). A fibronectin extra domain B (EDB)-specific aptide (APTEDB) was used as a cancer-specific targeting ligand. An APTEDB-DTX conjugate was synthesized from an alkyne-modified aptide and azide-modified DTX via click chemistry. A microscopy study revealed selective binding of dye-labeled APTEDB to EDB-overexpressing cancer cells. The cytotoxicity of the conjugate toward EDB-overexpressing murine lung carcinoma (LLC) and human glioblastoma (U87MG) was similar to that of free DTX. In a pharmacokinetic study, APTEDB-DTX formulated with PEG400/ethanol(5%) exhibited a circulation half-life similar to that of a Tween-80/ethanol formulation of parent DTX. Finally, an evaluation of intravenously injected APTEDB-DTX in mice bearing EDB-positive tumors showed that APTEDB-DTX inhibited the growth of both LLC allograft and U87MG xenograft tumors with an efficacy better than the parent-DTX formulation but with much lower toxicity, as evidenced by reduced body weight loss. Taken together, these results indicate that the aptide-drug conjugate system described here may hold potential as a targeted therapy regimen.