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Biodiesel, derived from the transesterification of vegetable oils or animal fats with simple alcohols, has attracted more and more attention recently. As a cleaner burning diesel alternative, biodiesel has many attractive features including: biodegradability, non-toxicity, renewability and low emission profiles. Although cottonseed oil was the first commercial cooking oil in the U.S, it has progressively lost its market share to some vegetable oils that have larger production and less cost. However, regarding the active researches on biodiesel production from vegetable oils, there is a promising prospective for the cottonseed oil as a feedstock for biodiesel production, which may enhance the viability of the cottonseed industry. The focus of this research is to optimize the biodiesel production from crude cottonseed oil. The effect of variables including methanol/oil molar ratio, catalyst concentration, reaction time, reaction temperature, and rate of mixing on the biodiesel yield was examined and optimized by response surface methodology (RSM). Besides,