Paclitaxel (PTX) is one of the most effective chemotherapeutic drugs for the treatment of a variety of cancers. However, it is associated with serious side effects caused by PTX itself and the Cremophor EL emulsifier. In the present study, we report the development of a well-defined amphiphilic linear-dendritic copolymer (named as telodendrimer) composed of polyethylene glycol (PEG), cholic acid (CA, a facial amphiphilic molecule) and lysine, which can form drug-loaded core/shell micelles when mixed with hydrophobic drug, such as PTX, under aqueous condition. We have used PEG(5k)-CA(8), a representive telodendrimer, to prepare paclitaxel-loaded nanoparticles (PTX-PEG(5k)-CA(8) NPs) with high loading capacity (7.3 mg PTX/mL) and a size of 20-60 nm. This novel nanoformulation of PTX was found to exhibit similar in vitro cytotoxic activity against ovarian cancer cells as the free drug (Taxol) or paclitaxel/human serum albumin nanoaggregate (Abraxane). The maximum tolerated doses (MTDs) of PTX-PEG(5k)-CA(8) NPs after single dose and five consecutive daily doses in mice were approximately 75 and 45 mg PTX/kg, respectively, which were 2.5-fold higher than those of Taxol. In both subcutaneous and orthotopic intraperitoneal murine models of ovarian cancer, PTX-PEG(5k)-CA(8) NPs achieved superior toxicity profiles and anti-tumor effects compared to Taxol and Abraxane at equivalent PTX doses, which were attributed to their preferential tumor accumulation, and deep penetration into tumor tissue, as confirmed by near infrared fluorescence (NIRF) imaging.