Abstract Background All-trans-retinoic-acid (ATRA) is a promising agent in the prevention/treatment of breast-cancer. There is growing evidence that reprogramming of cellular lipid metabolism contributes to malignant transformation and progression. Lipid metabolism is implicated in cell differentiation and metastatic colonization and it is involved in the mechanisms of sensitivity/resistance to different anti-tumor agents. The role played by lipids in the anti-tumor activity of ATRA has never been studied. Methods We used 16 breast cancer cell-lines whose degree of sensitivity to the anti-proliferative action of ATRA is known. We implemented a non-oriented mass-spectrometry based approach to define the lipidomic profiles of each cell-line grown under basal conditions and following treatment with ATRA. To complement the lipidomic data, untreated and retinoid treated cell-lines were also subjected to RNA-sequencing to define the perturbations afforded by ATRA on the whole-genome gene-expression profiles. The number and functional activity of mitochondria were determined in selected ATRA-sensitive and –resistant cell-lines. Bio-computing approaches were used to analyse the high-throughput lipidomic and transcriptomic data. Results ATRA perturbs the homeostasis of numerous lipids and the most relevant effects are observed on cardiolipins, which are located in the mitochondrial inner membranes and play a role in oxidative-phosphorylation. ATRA reduces the amounts of cardiolipins and the effect is associated with the growth-inhibitory activity of the retinoid. Down-regulation of cardiolipins is due to a reduction of mitochondria, which is caused by an ATRA-dependent decrease in the expression of nuclear genes encoding mitochondrial proteins. This demonstrates that ATRA anti-tumor activity is due to a decrease in the amounts of mitochondria causing deficits in the respiration/energy-balance of breast-cancer cells. Conclusions The observation that ATRA anti-proliferative activity is caused by a reduction in the respiration and energy balance of the tumor cells has important ramifications for the therapeutic action of ATRA in breast cancer. The study may open the way to the development of rational therapeutic combinations based on the use of ATRA and anti-tumor agents targeting the mitochondria.