The influence of surface charge on biodistribution and tumor accumulation remains debatable because most research has been carried out by changing the surface functional groups of nanocarriers. In this work, to avoid the interference of different surface properties such as chemical composition and hydrophilicity, polymeric micelles with uniform PEG coatings and continuously tunable sizes or zeta potentials were developed via a facile route. Therefore, the influence of surface charge on the biological functions of micelles with the same size and surface properties could be well explored. In this case, positive charge was found to enhance both tumor cellular uptake and tumor accumulation. Immunofluorescence staining indicated that the improved tumor accumulation was mainly due to the tumor vasculature targeting of positively charged micelles. It is predicted that efficient drug delivery systems for both tumor vasculature and cancer cell targeting can be realized based on positively charged micelles.