A statistical approach was used for optimization of the transesterification reaction from cottonseed oil, and the effects of five-level-four-factors and their reciprocal interactions were estimated. Thirty individual trials were designed in this experiment. The reaction temperature, methanol/oil molar ratio, catalyst amount, and reaction time were optimized using a central composite design. A statistical model predicted that the highest conversion yield of biodiesel from cottonseed oil would be 98.67%, at the following optimized conditions: the reaction temperature is 65 degrees C, the methanol/oil molar ratio is 7:1, the catalyst amount is 1.0 wt%, and the reaction time is 60 min. The reaction temperature is the most important factor, having a positive influence on conversion yield of biodiesel. The six independent experiments were replicated under experimental conditions using these optimal factor values, and an average content of 97.8 +/- 0.6% was achieved. This value was well within the range predicted by the model.