A close interaction between mitochondria and the cytoskeleton is essential to ensure the proper distribution of mitochondria within a cell. Recent studies have highlighted interactions between intermediate filaments, notably NFL or Vimentin and the key molecules necessary for the maintenance of organelle integrity and mitochondrial motility. The NFL-TBS.40-63 peptide is able to alter microtubule formation when it is internalized by T98G glioblastoma cells and inhibits their proliferation. In this study, we have evaluated the effect of NFL-TBS.40-63 peptide internalization on mitochondrial biogenesis and function. Our observations revealed a negative impact on T98G cell respiration after 6 hours of NFL-TBS.40-63 peptide treatment. This action on mitochondrial function, at lower concentrations than those necessary to disturb the cytoskeleton, could be related to a primary modification of the mitochondrial motility. It has been shown that peptides derived from the N-terminal domain of intermediate filaments, like desmin, vimentin and keratin, can interact with the unpolymerized tubulin. A recent study demonstrated that the N-terminal domain of vimentin can also directly bind mitochondria and serve as an adaptor between actin microfilaments and mitochondria. We suggest that the primary action of NFL-TBS.40-63 leads to sequential organization of the peptide, which disturbs the cytoskeleton and reorganizes the mitochondrial network. This should be related to the conserved FIS1/MFN2 ratio we observed at higher peptide concentrations. At a 10 mM peptide treatment, we also revealed a colocalization of NFLTBS. 40-63 with mitochondria and a specific accumulation at the microtubules’ extremities, which may limit membrane ruffling, as previously reported. This study revealed that the NFL-TBS.40-63 peptide provokes a redistribution of mitochondria throughout the cytoplasm.