Abstract Serotonin (5-HT) can stimulate the cholinergic system of the uterus by indirect actions on the modulation of reflexes and a direct action on smooth muscles. This study aimed to investigate the role of 5-HT in the regulation of the cholinergic activity in the uterine parts of non-pregnant rabbits. The right vagus or pelvic nerve and the left sympathetic trunk were stimulated by an electrical field, and the uterine contractile activity was evaluated by measuring the amplitude and frequency of slow wave electromyogram (EMG), with the surface of microelectrodes applied to the uterus bottom, body, and cervix, respectively. Double stimulation of the vagus or pelvic nerve and the serotoninergic fibers of the sympathetic trunk increased the frequency and the amplitude of the slow wave EMG in all the uterine parts. Furthermore, the administration of exogenous 5-HT increased the vagus or pelvic induced EMG activity in all parts of the uterus. Overall our results demonstrate that 5-HT enhances the vagus contractile activity with a magnitude of the effect decreasing from the bottom to the cervix, whereas 5-HT enhances the pelvic nerve contractile functions with a magnitude of the response increasing from the bottom to the cervix. The administration of droperidol, a 5-HT3 and 4 receptor inhibitor, and spiperone, a 5-HT2 receptor antagonist, inhibited the effect of the serotoninergic fibers of the sympathetic trunk to increase the vagus and pelvic nerve EMG activity. These data suggest that 5-HT stimulation of the parasympathetic nerves results in the induction of uterine contraction via the activation of 5-HT2, 3, and 4 receptor subfamilies.