DuringMycobacterium tuberculosis (Mtb)infection, the virulence factor PtpA belonging to the protein tyrosine phosphatase family is delivered into the cytosol of the macrophage. PtpA interacts with numerous eukaryotic proteins modulating phagosome maturation, innate immune response, apoptosis, and potentially host-lipid metabolism, as previously reported by our group.In vitro, the human trifunctional protein enzyme (hTFP) is abona fidePtpA substrate, a key enzyme of mitochondrial β-oxidation of long-chain fatty acids, containing two alpha and two beta subunits arranged in a tetramer structure. Interestingly, it has been described that the alpha subunit ofhTFP (ECHA,hTFPα) is no longer detected in mitochondria during macrophage infection with the virulentMtbH37Rv. To better understand if PtpA could be the bacterial factor responsible for this effect, in the present work, we studied in-depth the PtpA activity and interaction withhTFPα. With this aim, we performed docking andin vitrodephosphorylation assays defining the P-Tyr-271 as the potential target of mycobacterial PtpA, a residue located in the helix-10 ofhTFPα, previously described as relevant for its mitochondrial membrane localization and activity. Phylogenetic analysis showed that Tyr-271 is absent in TFPαof bacteria and is present in more complex eukaryotic organisms. These results suggest that this residue is a specific PtpA target, and its phosphorylation state is a way of regulating its subcellular localization. We also showed that phosphorylation of Tyr-271 can be catalyzed by Jak kinase. In addition, we found by molecular dynamics that PtpA andhTFPαform a stable protein complex through the PtpA active site, and we determined the dissociation equilibrium constant. Finally, a detailed study of PtpA interaction with ubiquitin, a reported PtpA activator, showed that additional factors are required to explain a ubiquitin-mediated activation of PtpA. Altogether, our results provide further evidence supporting that PtpA could be the bacterial factor that dephosphorylateshTFPαduring infection, potentially affecting its mitochondrial localization or β-oxidation activity.