Organophosphorus (OP) insecticides are used worldwide to protect agricultural crops and dwellings. These chemicals phosphorylate diverse serine hydrolases, including acetylcholinesterase. Among them, fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), components of the endocannabinoid signaling system (ECS) in male reproductive organs, are candidate targets of OP insecticide-induced spermatotoxicity. The effects of OP insecticides on the ECS in male reproductive accessory organs have not yet been investigated. In the present study, we examined the potential inhibitory effect of dichlorvos (DDVP) against FAAH and MAGL in male reproductive organs. In vitro screening assays were conducted by activity-based protein profiling with a fluorophosphonate chemical probe using samples of the testis, epididymis, prostate, and seminal vesicle of Wistar rats. Ex vivo assays were then performed using organs from rats orally administered 0, 5, or 10 mg/kg DDVP, 6 days per week for 9 weeks. In vitro assays showed that DDVP inhibited FAAH in the proteomes of rat testis, epididymis, and prostate, but scarcely inhibited MAGL. DDVP failed to inhibit FAAH and MAGL in the seminal vesicles. Ex vivo assays confirmed inhibition of FAAH in the proteomes of the prostate, testis, and epididymis of DDVP-treated rats, which exhibited morphologically abnormal sperm and decreased sperm motility. In conclusion, DDVP reduced the activity of FAAH but not of MAGL in male reproductive organs excluding the seminal vesicle; prostate involvement was demonstrated for the first time. Endocannabinoid signaling inhibition in these organs might contribute to sperm abnormality via deterioration in seminal plasma quality.