The autonomic efferent neurotransmission to the bladder and prostate smooth muscle is a potential target for drug therapy of specific low urinary tract disfunction (LUTD). Since amikacin and other amynoglicosides were reported to affect neurotransmission by a pre-junctional mechanism, we investigated the effect of amikacin on isolated rat and human detrusor smooth muscle contraction and on isolated rat and human prostate contraction, to further evaluate its potential relaxant properties. Materials and Methods Samples of detrusor smooth muscle and prostate tissue, obtained from 97 rats and 16 patients undergoing surgery, were studied through the measurement of isometric contraction induced by electrical field stimulation (EFS) and other pharmacological stimuli in the presence or absence of 1mM amikacin in a low-Ca medium. Results Amikacin 1 mM significantly reduced contraction of isolated rat and human detrusor muscle and prostate, achieved with pre-junctional stimulation, while no significant effect was observed on contraction induced by pharmacological post-junctional stimulators. EFS contraction inhibited by amikacin was restored after addition of calcium chloride. The amikacin effect was comparable to the effect of magnesium ions, which are known to exert a pre-junctional inhibition of neurotransmitter release. Conclusions Amikacin significantly inhibited rat and human detrusor and prostate contraction evoked by pre-junctional stimulation in vitro, suggesting a depressant effect on autonomic efferent neurotransmission. Further pharmacokinetics studies and researches on related compounds may hold potential for future development in the treatment of specific low urinary tract disfunction (LUTD).