In folklore medicine Ananas comosus (pineapple) is reputed to act as an abortifacient and in expectant women as a means of inducing labor.Several reports have claimed abortifacient property of A. comosus fruit (ripe or unripe). Ripe fruit has been used orally as traditional medicine in inducing abortion in Kerala state of India while the juice of unripe fruit was used for abortion in Bangladesh. However, scientific evidence supporting the efficacy of pineapple extracts in inducing uterine contractions is clearly lacking.This study investigated the pharmacological effects of different fractions of pineapple extract with a range of maturities to identify the most potent uterotonic fraction.The ethanolic crude extracts of pineapple (edible part) were prepared and fractionated through a series of liquid-liquid partitions. Fractions were separately tested on isolated uterine muscle from pregnant SD rats and human pregnant myometrium, which were cut into strips along the longitudinal axis of uterus. The strips were mounted vertically in organ baths (37 °C) and exposed to cumulative addition of fractions (0.1–10 mg ml−1), serotonin (0.05–5 µM) and different inhibitors to delineate the mechanism of action of the active ingredients of the extract.Aqueous fraction (F4) possesses uterine stimulant property which was blocked by verapamil but unaffected by indomethacin, prazosin and atosiban. Notably, ketanserin (10 µM) diminished the maximal contractile response induced by both F4 and 5HT by 74.3% and 92.1% respectively.These results may indicate the presence of 5HT or 5HT-like compound(s) and serotonergic pathways may contribute to the uterotonic activity of pineapple extract.