A new class of mixed-charged zwitterionic copolymer poly(aminoethyl methacrylate)- co-poly(methacrylic acid)- co-poly( n-butyl methacrylate) (CPMA) was prepared as drug nanocarrier for efficient intracellular delivery of Doxorubicin (DOX). The mixed-charged CPMA copolymer could readily assemble to micelles in physiological environment (pH 7.4) with the size of 42.6 nm and zeta potential of -26 mV, which would lead to a prolonged circulation time and enhanced tumor penetration. However, the micelles formed large aggregates due to the protonation of carboxyl groups at extracellular tumor pH (pH 6.5). Meanwhile, the zeta potential of CPMA micelles increased from -26 mV to -6 mV when the solution pH was changed from pH 7.4 to pH 6.5. The increase of size and zeta potential at extracellular tumor pH could benefit the retention of micelles in tumor matrix and uptake by cancer cells. The DOX-loaded mixed-charged CPMA micelles could induce a higher internalization at pH 6.5 than 7.4 at varied time periods. Moreover, cytotoxicity assay demonstrated that the blank micelles showed excellent biocompatibility, but were highly cytotoxic toward KB cells after loading with DOX. Thus, the mixed-charged zwitterionic polymeric micelles might be a promising carrier for tumor acidic environment responsive drug delivery.