© 2019 Elsevier B.V. Paclitaxel (PTX), a water insoluble anticancer drug, was incorporated into the inner aqueous core of a liposome without the aid of an organic co-solvent, via non-covalent binding with bovine serum albumin (BSA) to form a PTX-BSA liposome. In the present study, PTX-BSA-liposomes are shown to have potent effects on human-derived breast cancer cell lines, MCF-7 cells and MDA-MB-231 cells, in 2D monolayer cultured cells and 3D multicellular tumor spheroids. The results of cellular uptake studies in 2D monolayer cultured cells clearly showed that the fluorescence derived from dansyl-L-asparagine (DNSA), a model encapsulated drug, and Cy5-cholesterol (a model membrane) of DNSA-BSA-liposome were observed inside the cells. Along with cell uptake, the PTX-BSA-liposomes exhibited a concentration-dependent cytotoxicity against MCF-7 and MDA-MB-231 cells but the IC50 value of the PTX-BSA-liposomes was higher than that of free PTX and nab-PTX (albumin-bound PTX nanoparticle). On the other hand, PTX-BSA-liposome, as in the cases of free PTX and nab-PTX, inhibited cell growth in both 3D MCF-7 and MDA-MB-231 tumor spheroids, indicating that PTX-BSA-liposomes penetrated into the tumor spheroid. These results suggest that PTX-BSA-liposomes are an organic solvent free PTX formulation that would have potent anti-proliferative effects against breast cancer.