Due to preferential electrostatic adsorption of multivalent cations on highly anionic surfaces, natural multivalent polyamines and especially tetravalent spermine can be considered as potential regulators of the complex dynamical properties of anionic microtubules (MTs). Indeed the C-terminal tails of tubulin display many negative residues in a raw which should enable the formation of a correlated liquid-like phase of multivalent counterions on its surface. Although it is known that polyamine counterions promote MT assembly in vitro, little is known about the relevance of this interaction in vivo. We explore here the relationship between polyamine levels and MT assembly in HeLa and epithelial NRK cells using DFMO, an irreversible inhibitor of ornithine decarboxylase, and APCHA, a spermine synthase inhibitor. Under conditions of intracellular polyamine depletion, the MT network is clearly disrupted and the MT mass decreases. Addition of spermine to polyamine-depleted cells reverses this phenotype and rapidly promotes the extensions of the MT network. Finally we show that polyamine levels modulate the coating of MTs with MAP4, a MT-stabilizing protein, and the spatial distribution of EB1, a MT plus-end binding protein. In addition, polyamines favor the formation of gap junctions in NRK cells, a process which requires MT extensions at the cell periphery. This work provides basis for a better understanding of the role played by polyamines in MT assembly and establishes polyamine metabolism as a potential cellular target for modulating MT functions.