Abstract: The production of biodiesel and nontoxic cottonseed meal by in-situ alkaline-catalyzed transesterification of the triglycerides (TG) in the cottonseed was investigated. The effect of the moisture content and particle size of cottonseed flour on the fatty acid methyl ester (FAME) content in the biodiesel and free gossypol (FG) content in the cottonseed meal was investigated. The reaction conditions including the molar ratio of methanol to oil, amount of alkaline catalyst used, and reaction temperature and time were optimized by the mono-factor and orthogonal tests. When the cottonseed flour with the particle size of less than 0.335mm and the moisture content of appreciate 1.92% is used, the FAME and FG contents achieve 97% and 0.031%, respectively, under the operation conditions of 30℃, methanol/oil mol ratio being 135, 0.10mol/L NaOH in methanol, and reaction for 3h. The FG content in the cottonseed meal obtained from in-situ alkaline-catalyzed transesterification is below the FAO standard.