Epithelial to mesenchymal transition (EMT) is a biological process that plays a crucial role in cancer metastasis. Although studies regarding the EMT mechanisms are usual in terms of gene expression and protein functions, little is known about the involvement of lipids in EMT. In this work, an untargeted lipidomic analysis was performed to reveal which lipids are involved in the EMT process. DU145 prostate cancer cells were treated with TNFα, a well-known EMT inducer. After 6 hours of treatment, a decrease of cell membrane E-cadherin as well as a reduction in its gene expression were observed. Also, the mesenchymal markers Vimentin and Snail were up-regulated, suggesting that EMT started below 6 hours of treatment. Lipid extracts of untreated and TNFα-treated cells at short times were analyzed using ultra-performance liquid chromatography coupled to high-resolution mass spectrometry (UPLC-MS). Multivariate data analysis methods were applied to decipher which lipids presented significant changes after EMT induction. Among the results obtained, a significant increase of twelve unsaturated triacylglycerides (TAGs) was observed. This increase of TAGs was also observed for cells treated with TGFβ (another EMT inducer), suggesting that this feature is a common mechanism in the EMT process. In conclusion, this work reported for the first time a TAG accumulation through EMT induction. These TAG lipids could play a key role in providing cells with the energy, cell membrane components and signaling lipids necessary to guarantee the enhanced cell migration and proliferation of metastatic cells.