Through a series of in vitro studies, the essential steps for intracellular drug delivery of paclitaxel using a pH-responsive nanoparticle system have been investigated in breast cancer cells. We successfully encapsulated paclitaxel within polymeric expansile nanoparticles (Pax-eNPs) at 5% loading via a miniemulsion polymerization procedure. Fluorescently tagged eNPs were readily taken up by MDA-MB-231 breast cancer cells grown in culture as confirmed by confocal microscopy and flow cytometry. The ability of the encapsulated paclitaxel to reach the cytoplasm was also observed using confocal microscopy and fluorescently labeled paclitaxel. Pax-eNPs were shown to be efficacious against three in vitro human breast adenocarcinoma cell lines (MDA-MB-231, MCF-7, and SK-BR-3) as well as cells isolated from the pleural effusions of two different breast cancer patients. Lastly, macropinocytosis was identified as the major cellular pathway responsible for eNP uptake, as confirmed using temperature-sensitive metabolic reduction, pharmacologic inhibitors, and fluid-phase marker colocalization.