Oxytocin is a peptide hormone that mainly functions to control the contractility of smooth muscles and sex‐related steroidogenesis in male reproductive tracts. However, specific information concerning this hormone in controlling the reproductive organs of cats is limited. This study aimed to investigate the expression of oxytocin receptors (OTRs) and their signal mediator via prostacyclin synthase (PTGIS) in reproductive structures following oxytocin assisted electroejaculation. In Experiment 1, the testis, cauda epididymis and vas deferens from five cats were examined by immunohistochemistry and quantitative polymerase chain reaction in order to study the responses of OTR and PTGIS mRNA to oxytocin injection. Experiment 2 examined the effect of oxytocin administration prior to electroejaculation on ejaculate characteristics and sperm quality in terms of motility, viability and fertilizing ability. Immunohistochemistry revealed the expression of OTRs in Leydig's, peritubular myoid cells and some spermatogenic cells. The expression was found in the epithelium and smooth muscle of the epididymis and vas deferens. After oxytocin administration, the OTR mRNA was upregulated in the epididymis (p > .05) and vas deferens (p = .01). The expression level of PTGIS mRNA increased in the response to oxytocin treatment only for the vas deferens (p > .05). Oxytocin treatment before electroejaculation resulted in an approximately twofold increase in sperm concentration and total sperm output/ejaculate, while this intervention did not significantly affect ejaculate volume, sperm quality or fertilizing ability. This study concluded that the oxytocin cascade is locally present in the reproductive structures and plays a role in promoting sperm delivery during electroejaculation in cats.