The endocannabinoid system and prostaglandins are important modulators in the genitourinary system. This study aimed to investigate the possible interactions between the endocannabinoid system and the cyclooxygenase (COX) pathway on rat vas deferens. For this purpose, the concentration responses of the endocannabinoid anandamide, prostaglandin F 2α analog latanoprost, and prostaglandin E 1 analog misoprostol on the electrical field stimulation (EFS)-induced contractile responses were obtained. The concentration responses to anandamide were obtained again in the presence of nonselective COX inhibitor flurbiprofen and prostaglandin analogs, while the concentration responses of latanoprost and misoprostol were obtained in the presence of cannabinoid receptor antagonists and fatty acid amide hydrolase (FAAH) enzyme inhibitor URB597. FAAH, COX-1, and COX-2 enzyme levels in vas deferens tissue samples were also determined. The cumulative addition of anandamide was not different from the vehicle; however, the EFS-induced contractile responses were significantly increased with the incubation of latanoprost or flurbiprofen in the prostatic portion. Flurbiprofen and misoprostol decreased FAAH enzyme levels in both portions of the vas deferens, while latanoprost induced the inhibition in the prostatic portion. The cumulative administration of latanoprost and misoprostol significantly enhanced the contractile responses in the prostatic portion. This effect of latanoprost was significantly antagonized by URB597 and AM251. The enhancing effect of misoprostol was antagonized by anandamide, URB597, AM251, and AM630. Anandamide, AM251, AM630, and URB597 decreased enzyme levels of COX-1 and COX-2 in both portions of the vas deferens. These results demonstrate an intricate crosstalk between endocannabinoids and prostaglandins in modulation of the vas deferens contractility.