According to studies, adult men with high urine levels of the over-the-counter mild painkiller APAP had decreased sperm mobility and a longer time to conception. The study included an in vivo human pharmaceutical APAP exposure experiment to determine the extent to which APAP reached sperm cells in the seminal fluid; in vitro calcium imaging and functional experiments in freshly donated human sperm cells to investigate CatSper channel-dependent activation by APAP and its metabolites; and experiments to determine the in situ capabilities of human sperm cells to form APAP metabolite AM404. Consenting beforehand, 3 healthy young men participated in the in vivo human exposure experiment. On the day of the in vitro experiments, samples of human sperm were donated by young, healthy volunteers who consented beforehand. High micromolar quantities of pharmaceutical APAP reached the seminal plasma and accumulated in the seminal plasma between 3 and 5 days after exposure (P-value 0.023). APAP and its major metabolite 4-aminophenol (4AP) do not affect Ca2+– signaling in human sperm. Instead, the APAP metabolite AM404 generated by FAAH interferes with Ca2+– signaling in human sperm via a CatSper-dependent mechanism. AM404 greatly enhances sperm cell penetration through viscous mucus (P-value of 0.003). FAAH was functionally expressed in the neck/midpiece area of human sperm cells, as demonstrated by immunohistochemical labeling and the ability of human sperm cells to hydrolyze the FAAH substrate arachidonyl 7-amino, 4-methyl coumarin amide in a FAAH-dependent manner. Importantly, human sperm cells could produce AM404 in situ after exposure to 4AP (P-value 0.0402 compared to vehicle-treated sperm cells). The majority of the trials were conducted in vitro. Future research was required to determine whether APAP could inhibit human sperm activity in vivo via AM404.