The development of effective oral preparations of 2-methoxyestradiol (2-ME) has been a worldwide problem. In this study, breakthrough progress has been made in oral delivery of 2-ME by encapsulating 2-ME micelles in pH-responsive microspheres. 2-ME micelles with small particle size (58 nm) and high drug loading (7.94 ± 0.23%) were successfully prepared and showed slower release characteristics and 12.8 times higher cytotoxicity on 4T1 cells than free 2-ME. The pH-sensitive microspheres could slowly release 82% of 2-ME micelles for 10 h in PBS (7.4). After oral administration to rats, 2-ME-micelles-microspheres compared to 2-ME micelles could prolong significantly blood retention time (MRT) of 2-ME by 12 times and enhance oral absolute bioavailability from 49.99% to 121.68%. In vivo imaging of 4T1 tumor-bearing mice showed that retention time and intensity of the fluorescence signal in each 2-ME preparation group all were consistent with the pharmacokinetic results and intact 2-ME micelles in the microspheres could be absorbed in intestine to blood to target to tumor. Furthermore, after the 4T1 tumor-bearing mice were treated with oral 2-ME-micelles-microspheres for 21 days, the tumor growth inhibition rate achieved 94.93% and the mice weight gained significantly, indicating their high antitumor activity and low toxicity. While oral administration of 2-ME micelles and 2-ME microspheres all were ineffective under the same conditions. Therefore, micelles-microspheres might be a promising candidate for oral administration of 2-ME for cancer therapy.