Tanshinone IIA (TAN) has few clinical applications for anti-cancer therapy mainly due to its high lipophicity, low cellular uptake, and poor bioavailability. To improve the anti-cancer effect and bioavailability of TAN, we developed a mixed micelle system constituted with d-α-tocopheryl polyethylene glycol succinate-graft-poly(d,l-lactide-co-glycolide) (TPGS-g-PLGA) copolymer and Pluronic F68. TAN was encapsulated in the TPGS-g-PLGA/Pluronic F68 mixed micelles by using the thin film hydration technology optimized by the central composite design/response surface method (CCD/RSM). TAN-loaded mixed micelles were highly stable in the presence or absence of bovine serum albumin (BSA) and achieved sustained drug release in vitro. Compared with free TAN, TAN mixed micelles had higher cytotoxicity and pro-apoptotic effects against human hepatocellular carcinoma HepG2 cells. The significant enhancement on pro-apoptosis by TAN micelles was evidenced by increased chromosome condensation, mitochondria membrane potential loss, cell apoptosis, and cleavages of caspase-3 and PARP. Furthermore, pharmacokinetic studies revealed that TAN mixed micelles significantly prolonged the circulation time and improved bioavailability of TAN in rats. These results demonstrated that TAN-loaded TPGS-g-PLGA/F68 mixed micelles are an effective strategy to deliver TAN for cancer therapy. © 2014 Elsevier B.V. All rights reserved.