Cancer is one of the main causes of death in the world and thus a global public health problem. Among the treatments available for cancer are surgery, radiotherapy, and chemotherapy. Currently, there is increased interest in the combination of two or more antitumor agents to achieve a synergistic effect in cancer therapy. Doxorubicin (DOX), a chemotherapeutic which has a potent antineoplastic action, has been used in the treatment of various tumors. However, the use of DOX is limited, mainly due to the cardiotoxicity. Therefore, nanostructured systems, such as liposomes, have been developed to carry this drug and target the tumor region, since tumor tissues present enhanced permeability and retention for nanosystems. Cardiac glycosides, such as digitoxin, have recently shown great antitumor potential despite the low therapeutic index which may limit their use. Furthermore, some compounds of this class have low water solubility, which makes their in vivo administration difficult. In this context, liposomes represent a valid strategy to carry simultaneously antitumor drugs allowing their intravenous administration. In this study, liposomes loaded with glucoevatromonoside containing peracetylated glucose hydroxyl groups (GEVPG) and DOX at molar ratio of 1:1 (SpHL-GEVPG:DOX 1:1) were developed, and their chemical and physicochemical properties were evaluated. This formulation presented a combination index (CI) lower than 1 at inhibitory concentration of 90 % growth (IC₉₀) for three human breast tumor lines evaluated (0.52 ± 0.39 for MDA-MB-231, 0.19 ± 0.13 for MCF-7, and 0.99 ± 0.09 for SKBR-3). These results indicate a synergistic cytotoxic effect of the GEVPG and DOX combination encapsulated in liposomes. In addition, SpHL-GEVPG:DOX 1:1 presented selectivity towards these cancer cells. Long-term in vitro cytotoxicity studies demonstrated that MDA-MB-231 surviving cells after treatment with SpHL-GEVPG:DOX 1:1 did not recover proliferation capacity after 21 d. From the studies of cell cycle and death pathway evaluation, it was observed that SpHL-GEVPG:DOX 1:1 arrested the cell cycle in the G2/M phase and similarly induced apoptosis and necrosis. However, SpHL-GEVPG:DOX at molar ratio of 1:1 showed lower induction of both apoptotic and necrotic pathways compared to free DOX and SpHL-DOX, suggesting that the mechanism of death involved may not be related to necrosis or apoptosis. Lastly, SpHL-GEVPG:DOX 1:1 showed a good storage stability for 90 d at 4 °C. Therefore, the results of the present work indicate the potential use of SpHL-GEVPG:DOX 1:1 as a new anticancer formulation.