Sexual experience in male rodents, induced by a first exposure to a receptive female, improves efficiency of following copulations. In mice, the mechanisms supporting this improvement are poorly understood. We characterized molecular modifications of the mouse hypothalamic medial preoptic area (mPOA), the main integrative structure for male sexual behaviour, after a single mating event. This paradigm induced long-lasting behavioural improvements and mPOA morphological changes, evidenced by dendritic spine maturation and an increase in the acetylated and tri-methylated forms of histone H3. Ejaculation affected testosterone, progesterone and corticosterone levels in both naive and experienced mice, but sexual experience did not modify basal plasma or hypothalamic levels of steroids. In contrast to studies carried out in rats, no changes were observed, either in the nitrergic system, or in sex steroid receptor levels. However, levels of glutamate- and calcium-associated proteins, including PSD-95, calbindin and the GluN1 subunit of the NMDA receptor, were increased in sexually experienced male mice. The Iba-1 microglial marker was up-regulated in these animals suggesting multicellular interactions induced within the mPOA by sexual experience. In conclusion, plasticity mechanisms induced by sexual experience differ between rat and mouse, even if in both cases they converge to potentiation of the mPOA network.