Pancreatic ductal adenocarcinoma (PDAC) tumour-initiating cells are a subpopulation of cancer cells displaying unique features such as increased chemoresistance and a metabolic phenotype promoting a higher metastatic potential. Therefore, a successful therapy must eradicate these cells to improve the poor outcome that PDAC patients face. Recent evidence suggests that metabolism of these cancer stem-like cells relies on oxidative phosphorylation and they are responsible for tumour relapse. Here we present a metabolic characterization of tumour-initiating cells derived from a panel of pancreatic cancer cell lines. Seahorse XF analysis has evidenced that, although some cells show increased OxPhos metabolism, results highlight a more complex metabolic profile when different cell lines are compared. This cell line-dependent complexity is also confirmed comparing the expression of cell surface markers using Flow Cytometry (CD44, CD24, EpCAM and CD133) between cancer cells and their corresponding tumour-initiating cells. When induced from their corresponding parental cancer cells, tumour-initiating cells display a distinctive spheroid-like morphology (tumourspheres) and western blot has evidenced the overexpression of a set of ATP-binding cassette transporters which are responsible for increased chemoresistance. In fact, dose response to chemotherapy drugs show that tumour-initiating cells are more resistant than their corresponding parental cancer cells. These data indicate that pancreatic cancer tumour-initiating cells represent a powerful approach to investigate novel metabolic targets for therapeutic intervention. However, therapeutic targeting of metabolism vulnerabilities in cancer stem-like cells needs to take into consideration the heterogeneity found in this study.