<i>Introduction:</i> Normal and abnormal bladder contractions are principally mediated by acetylcholine released from postganglionic parasympathetic nerves. Since amikacin was reported to affect neurotransmission by a prejunctional mechanism, we investigated the effect of amikacin on isolated detrusor smooth muscle contraction to further evaluate its potential relaxant properties. <i>Materials and Methods:</i> Detrusor smooth muscle obtained from 15 rats and 8 patients undergoing surgery were studied through measurement of isometric muscular contraction induced with electrical field stimulation (EFS) (10–60 Hz), carbachol (10^–7 to 10^–3<i>M</i>) and nicotine (10^–7 to 10^–3<i>M</i>) in the presence or absence of 1 m<i>M</i> amikacin in a low-Ca medium. <i>Results:</i> Amikacin (1 m<i>M</i>) significantly reduced EFS-induced contraction of isolated rat and human detrusor muscle by 33 ± 6.57% (p < 0.005) and 40 ± 1.14% (p < 0.001), respectively. Contraction was restored after addition of calcium chloride (1 m<i>M</i>). The effect of amikacin was comparable to that of magnesium ions. Rat and human detrusor contractile response to nicotine was inhibited by 70 ± 8.27% (p < 0.001) and 64 ± 14.09% (p < 0.01) after the addition of amikacin (1 m<i>M</i>), while no significant effect was observed on carbachol-induced stimulation. <i>Conclusion:</i> Amikacin significantly inhibited detrusor contraction evoked by prejunctional stimulation in vitro, suggesting a depressant effect on autonomic neurotransmission in urinary bladder.