The floral biology and pollination ecology of durian, Durio zibethinus, were determined in eight semi-wild trees in mixed-fruit orchards in southern Thailand during April-May 2003 and 2005. Flowers open fully at 16h00–16h30 and most androecia drop around 01h00. Anthers dehisce at 19h30–20h00 when the stigmata are already receptive. In a series of pollination experiments, fruit was set in all treatments within 10 d. The greatest pollination success occurred after hand-crossed (76.6%), open (54.4%) and emasculation pollination (53.3%). Consistently, hand-crossed (12.2%), emasculation (8.7%) and open pollination (5.1%) yielded a substantial fruit set 2 mo after the pollination experiments. Very low pollination success in facilitated autogamy suggests that most durian trees are highly self incompatible. No mature fruit was found after insect pollination and automatic autogamy. Fruit bats, especially Eonycteris spelaea, are the major pollinators of this durian although the giant honey bee (Apis dorsata) was the most frequent visitor to the flowers. Bats visited durian flowers at the rate of 26.1 (SD = 20.7) visits per inflorescence per night. Since this semi-wild durian depends on fruit bats as its pollinator, protecting fruit bat populations and their roosts is vital for the production of the durian fruit crop.