Gadodiamide (Gd-DTPA-BMA) is a gadolinium-based complex, used as a contrast agent in magnetic resonance imaging procedures. Recent studies revealed the capacity of this complex of inducing apoptosis in neoplastic cells. However, a great challenge for its use as an anticancer drug is low cellular internalization and drug delivery systems such as thermosensitive liposomes can be a strategy to overcome these limitations and increase anti-tumor efficacy. In this context, this study aimed to develop, characterize, and assess the cytotoxic activity and selectivity index of thermosensitive liposomes containing Gd-DTPA-BMA. Formulations were prepared by the lipid film hydration method and their physicochemical, morphological, and thermal properties were evaluated. Cell viability was performed 4T1/MDA-MB-231 tumor cells and WI-26 VA4 normal cells. Transmission electron microscopy analyses showed high electron density in the inner core of Gd-DTPA-BMA-loaded liposomes that can be attributed to the Gd-DTPA-BMA, since Gd-loaded nanosystems present low light transmittance and high electron density by this technique. The dynamic light scattering, differential scanning calorimetry, small-angle X-ray scattering, and in vitro release results confirm that the lipid composition was suitable since T_c (temperature of the transition Lβ → Lα) values are in accordance with those reported for thermosensitive liposomes used in the treatment of cancer. The cytotoxic studies against breast cancer cell lines demonstrated that the Gd-DTPA-BMA-loaded liposomes have higher cytotoxicity than free Gd-DTPA-BMA. Moreover, these liposomes presented minimal toxicity in a normal cell line when compared to the free Gd-DTPA-BMA. Therefore, the results of this study suggest that thermosensitive liposomes containing Gd-DTPA-BMA might be a new promising nanocarrier system for breast cancer treatment.