Abstract Biodiesel is a fuel from vegetable oil or animal fat, and is a promising substitute for petroleum-derived diesel. Transesterification is the most widely used method in biodiesel production. Eggshell is rich in calcium carbonate (CaCO₃), and when it is subjected to heat treatment it results in calcium oxide (CaO). CaO from eggshells was prepared at different calcination temperatures, and characterized by X-ray diffraction, thermogravimetric analysis (TGA) and scanning electron microscopy (SEM). The obtained CaO was used as a catalyst. All catalysts showed good stability and excellent morphology for biodiesel synthesis. Catalytic activity was evaluated by the methyl transesterification reaction of cotton oil for 3 h, 9:1 methanol:oil molar ratio, 3 wt% (catalyst/oil weight ratio) catalyst and 60°C. Biodiesels showed an ester content of 97.83%, 97.23% and 98.08%, obtained from calcined eggshell at 800°C, 900°C and 1000°C, respectively. Biodiesel quality was affected by the acidity of the cation exchange resin. The kinematic viscosity of biodiesel was in accordance with specification, except for the biodiesel obtained from the calcined catalyst at 1000°C. The CaO from eggshells obtained at different calcination temperatures is promising for biodiesel synthesis.