Fast-growing demand for automobile vehicles and depletion of fossil fuel forced the researchers to think for alternative fuel which can replace the diesel fuel. From this perspective, Punnai oil which is non-edible in nature is chosen as a feedstock for producing methyl ester. Punnai oil can be converted into biodiesel/methyl ester by transesterification process. From gas chromatography analysis it is found that biodiesel of Punnai oil contains linoleic, oleic and palmitic fatty acids. Presence of these fatty acids and in the Punnai oil biodiesel will enhance the combustion characteristics. To ascertain the suitability of Punnai oil biodiesel as a fuel for direct injection Diesel engine, the experimental work was carried out using a constant speed, four-stroke single-cylinder Diesel engine. Experimental results show that there is a decrease in brake thermal efficiency and an increase in NOx and CO2 emissions with increased concentration of biodiesel in the blend. Smoke, CO, and HC emissions were reduced significantly. At rated power, brake thermal efficiencies of diesel, B20, B40, B60, and B80 are 29.2%, 28.6%, 28.1%, 27.5%, and 27%, respectively, and NOx emissions are in the order of 1516 ppm, 1547 ppm, 1553 ppm, 1567 ppm, and 1631 ppm. Smoke emission for diesel fuel was 50% whereas for B20, B40, B60, and B80, smoke emissions were 48%, 45%, 44%, and 43%. The same trend was observed for hydrocarbon emissions. Combustion characteristics of B20 blend closely follow the trend of diesel fuel. The maximum cylinder pressure of diesel and B20 are 68.3 bar and 67 bar, respectively, and maxi-mum heat release rate of diesel and B20 are 56 kJ/m3 oCA and 54 kJ/m3 oCA, respectively.