Seminal plasma (SP) has been shown to modulate the female reproductive environment in mammalian species in which the ejaculate comes into direct contact with the endometrium, such as mice, pigs and horses. In contrast, cattle ejaculate in the vagina, and it is questionable whether the fluid portion reaches the uterus. A recent study from our group reported that mating to intact, but not vasectomised, bulls modifies the endometrium transcriptome. However, it is not clear whether these changes are driven by SP molecules that bind to sperm at the time of ejaculation or not. Thus, the aim of the present study was to determine whether ejaculated sperm, which are suspended in the secretions of the accessory sex glands (AGs) at ejaculation, elicit a different endometrial transcriptomic response than epididymal sperm, which have never been exposed to SP factors of AG origin. To this end, endometrial explants collected from heifers in oestrus were (co-)incubated alone (control), or with epididymal or ejaculated sperm, following which RNA-sequencing analysis was performed. Endometrial exposure to epididymal sperm resulted in 48 differentially expressed genes (DEGs) compared with control explants. Strikingly, explants exposed to ejaculated sperm exhibited no DEGs compared with control explants. However, incubation with ejaculated sperm resulted in 80 DEGs in comparison with epididymal sperm, involved in muscle contraction, extracellular matrix remodelling, and immune system pathways, among others. These data indicate that ejaculated and epididymal sperm induce a different response in the endometrium, likely due to the acquisition of SP components at the time of ejaculation.