<b>Abstract</b>— <p>The biogenic polyamines are well known to regulate cell wall permeability for antibiotics permeating the cell via porins. The effect of polyamines on the antibiotics transported by the non-porin route, such as rifampicin, has not been studied. In this work, the effects of intracellular putrescine, spermidine, and cadaverine on the efficiency of rifampicin accumulation, the bacterial susceptibility to rifampicin, the hydrophobicity of the cell surface, as well as the effect of polyamines on the expression of the <i>marRAB</i> operon were tested. None of the three polyamines under study affected the rate of rifampicin transport into the cell at the early stages (2 min). Under longer exposure (60 min), a protective effect of cadaverine was observed, since accumulation of rifampicin in cadaverine-free cells was higher compared to cadaverine-proficient ones. The absence of cadaverine in <i>Escherichia coli</i> cells increased their hydrophobicity. There was a direct relationship between the cell surface hydrophobicity and the efficiency of rifampicin accumulation. Polyamines themselves did not affect the expression of the <i>marRAB</i> operon, but modulated its expression induced by salicylate. Putrescine had no effect, spermidine decreased the expression level, and cadaverine increased it. Overall, polyamine biosynthesis plays a role in bacterial adaptation to rifampicin, as the strains unable to synthesize cadaverine or putrescine and spermidine were more sensitive than the wild-type strains. Cadaverine plays a special role in protecting against the effects of rifampicin; its intracellular concentration affects bacterial susceptibility to rifampicin.</p>