The aim of this study was to investigate the effects of gentamicin on cow myometrium in vitro. After myometrial samples was contracted with 0.062, 0.125 and 0.250 mg/ml gentamicin was applied and effects on peak amplitude and frequency of contractions were investigated by 10 minute intervals. The frequency of oxytocin-induced contractions was 10.125±1.54 and peak amplitude was founded 3.70±0.65. Gentamicin dose dependently inhibited both amplitude and frequency of oxytocin-induced contractions of myometrium. Gentamicin dose-dependently decreased the the frequency values to 7.25±1.34 (P<0.001), 4.06±0.74 (P<0.001) and 0.43±0.2 (P<0.001). The mean peak amplitude of gentamicin dose-dependently decreased the peak amplitude values to 2.37±0.52 (P<0.001), 1.06±0.40 (P<0.001) and 0.45±0.24 (P<0.001) respectively. In conclusion, it was found that gentamicin inhibited both amplitude and frequency of oxytocin-induced contractions of myometrium obtained from cows.