Mitochondria are essential organelles for the evolution of complex animals, thought to have appeared two billion years ago in the precursor of the eukaryotes. Indeed, they enable the production of energy, the prerequisite of life, via complete oxidization of glucose by O2 into CO2 and H2O through oxidative phosphorylation. As the major carbon fuel for the TCA cycle, pyruvate is an important metabolite for the cell and its metabolism is under tight regulation. Despite almost 40 years of extensive research, it is only two years ago that the molecular components of the mitochondrial pyruvate carrier (MPC) were identified. This breakthrough opened a new frame to better understand pyruvate metabolism and its implications in diseases related to an impaired mitochondrial transport of pyruvate. Moreover, it presents a huge potential to develop cures for diseases related to pyruvate metabolism. The present work aimed at providing further characterization, both molecular and functional, of the newly identified MPC. On the molecular level, we tried to establish a protocol for large-scale protein expression of the MPC complex, in order to subsequently purify it. Here we show that the yeast expression system followed by His-tag purification is not appropriate to purify neither the mammalian MPC, neither the native complex nor the proteins separately. We propose an alternative strategy using a bacterial system to express human MPC1 and MPC2 constructs cloned with a N-terminal GST-His tag, followed by two-step affinity protein purification. Concerning the functional characterization of the carrier, our results do not support any effect of MPC1 knockdown on cancer cell proliferation. However, recent studies are rather suggesting that its overexpression is critical for many tumor cells. Finally, we show a general tendency of down-regulation of both MPC1 mRNA and protein levels in hypoxic conditions, and provide a model for a differential MPC expression depending on the oxygen availability.