There is a need to seek non-conventional seed oil sources for biodiesel production due to issues such as supply and availability as well as food versus fuel. In this context, Milo (Thespesia populnea L.) seed oil was investigated for the first time as a potential non-conventional feedstock for preparation of biodiesel. This is also the first report of a biodiesel fuel produced from a feedstock containing cyclic fatty acids as T. populnea contains 8,9-methylene-8-heptadecenoic (malvalic) and smaller amounts of two cyclopropane fatty acids besides greater amounts of linoleic, oleic and palmitic acids. The crude oil extracted from T. populnea seed was transesterified under standard conditions with sodium methoxide as catalyst. Biodiesel derived from T. populnea seed oil exhibited fuel properties of density 880 kg m-3, kinematic viscosity 4.25 mm2/s; cetane number 59.8; flash point 176 °C; cloud point 9 °C; pour point 8 °C; cold filter plugging point 9 °C; sulfur content 11 mg kg-1; water content 150 mg kg-1; ash content 15 mg kg-1; and acid value as KOH 250 mg kg-1. The oxidative stability of 2.91 h would require the use of antioxidants to meet specifications in standards. Generally, most results compared well with ASTM D6751 and EN 14214 specifications. © 2011 Elsevier Ltd.