During mating, transfer of sperm is accompanied by sexual stimulation of the female that is critical for successful reproduction. This stimulation engages several sensory systems and triggers species-dependent changes in female physiology and social behavior, such as preparation of the uterus for pregnancy and induction of sexual satiety. How does the female brain recognize sexual stimulation during mating to mediate the subsequent changes in female internal state that are necessary for reproduction?Here, I studied the function of the mouse serotonin (5-HT) system, an important modulator of female sexual function. First, I used fiber photometry to measure population calcium activity of dorsal raphe (DR) 5-HT neurons in freely behaving female mice during mating. I showed that female DR 5-HT neuronal activity increased at ejaculation to an amplitude unequaled by activity during any other phases of mating. This DR 5-HT signal was independent of the female’s sexual experience and was observed in both naturally cycling and ovariectomized females. I investigated further the nature of the stimulus that causes female DR 5-HT activation. At ejaculation, two critical events take place inside the female vagina: formation of the penis cup—a mouse penis behavior that results from erectile function—and forceful expulsion of ejaculatory fluid. These events could provide sexual stimulation via mechano- or chemosensory receptors. Artificial inflation of a small balloon inside the vagina triggered DR 5-HT neuronal activation, suggesting that mechanical stimulation could be a major component of the DR 5-HT signal seen at ejaculation. To test this hypothesis during natural mating, I surgically modified the ejaculatory apparatus in males, such that they were unable to deliver intra-vaginal mechanical stimulation at ejaculation, while still being able to perform other aspects of mating behavior. The DR 5-HT signal in females mating with these modified males was disrupted, in agreement with the mechanical stimulation hypothesis. Taken together, this research sheds light on a critical part of the mechanism underlying ejaculation detection by the female DR 5-HT system and has implications for its role in female sexual function.