Nigella sativa L. (NS) from Ranunculaceae family is known as black cumin in Indonesia. The seed has been used as an aphrodisiac in ethnobotanical studies and reported to have pharmacological activities such as antihypertensive through the relaxant effect of vascular smooth muscles but the direct effect to the blood vessels of the corpus cavernosum is still unknown. The purpose of this study was to examine the response of NS seed extract on penile erection in vitro. NS seeds were macerated in ethanol solvent for three days in room temperature and repeated for two times. Penile erection responses was assessed using isolated rat corpus cavernosum in Krebs-Henseleit solution, temperature 37°C, pH 7.4, aerated with carbogen gas. After acclimation, corpus cavernosum was contracted with a phenylephrine solution. Ethanolic extract of NS seeds or control solution were given after reaching the plateu phase of the highest contraction. This study showed that the contraction response of the corpus cavernosum decreased after addition of NS extract and this action was increased with the addition of the extract concentration. This study concluded that NS seed ethanol extract affects the penile erection response directly through the relaxation of blood vessels in the corpus cavernosum.