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The use of biodiesel is rapidly expanding around the world, making it imperative to fully understand the impacts of biodiesel on the diesel engine combustion process and pollutant formation. Biodiesel is known as the mono-alkyl-esters of long chain fatty acids derived from renewable feedstocks, such as, vegetable oils or animal fats, for use in compression ignition engines. Different parameters for the optimization of biodiesel production were investigated in the first phase of this study, while in the next phase of the study performance test of a diesel engine with neat diesel fuel and biodiesel mixtures were carried out. Biodiesel was made by the well known transesterification process. Cottonseed oil (CSO) was selected for biodiesel production. Cottonseed is non-edible oil, thus food versus fuel conflict will not arise if this is used for biodiesel production. The transesterification results showed that with the variation of catalyst, methanol or ethanol, variation of biodiesel production was realized. However, the optimum conditions for biodiesel production are suggested in this paper. A maximum of 77% biodiesel was produced with 20% methanol in presence of 0.5% sodium hydroxide. The engine experimental results showed that exhaust emissions including carbon monoxide (CO) particulate matter (PM) and smoke emissions were reduced for all biodiesel mixtures. However, a slight increase in oxides of nitrogen (NO
x
) emission was experienced for biodiesel mixtures.