Synchronized activation of autonomic and somatic divisions of the nervous system respectively destined to the seminal tract, including the bladder neck and the pelvi-perineal striated musculature, is necessary for anterograde ejaculation. We aimed at investigating the role of intermesenteric nerves (IMNs) in ejaculation in anesthetized rats. Electrical stimulation of intact IMNs and distal and proximal stumps of the sectioned IMN were tested in isoflurane-anesthetized male rats. Electrical stimulation of the intact IMN was also applied to rats with acute spinal transection at the T8 level. The effects of IMN electrical stimulation on emission and expulsion phases of ejaculation were evaluated by measuring seminal vesicle pressure (SVP) and bulbospongiosus (BS) muscle contractions, respectively. IMN electrical stimulation could induce SVP increase and rhythmic contractions of BS muscle concomitantly with expulsion of the seminal plug. When compared with intact IMN electrical stimulation, the occurrence of ejaculation and rhythmic BS muscle contractions, but not SVP increase, was reduced in response to electrical stimulation of the distal stump of the sectioned IMN. In comparison to intact IMN electrical stimulation, the occurrence of ejaculation and rhythmic BS muscle contractions was not significantly modified, whereas the increase in SVP was diminished when the proximal stump of the sectioned IMN was stimulated. Spinalization abolished ejaculation and rhythmic BS muscle contraction but did not impair SVP increase. It is concluded that both afferents conveyed by IMN and relaying supraspinally and efferents of IMN are involved in IMN electrical stimulation-induced ejaculation. We propose that the IMN electrical stimulation paradigm can be used to investigate physiological and pharmacologic aspects of ejaculation.