An experimental investigation was performed to analyze the effects of undiluted biodiesel fuel (100% methyl ester of soybean oil) on spray, combustion, and exhaust emissions characteristics in a direct injection (131) common-rail diesel engine with a cooled-EGR system. The macroscopic and microscopic injection characteristics for test fuels such as injection mass, injection rate, droplet diameter, and spray tip penetration were measured by using an injection rate meter, a spray visualization system, and a droplet measuring system. The study of combustion and exhaust emission characteristics was carried out under various engine operating conditions for biodiesel fuel, and the results were compared to those of ultra low sulfur diesel (ULSD). In this work, the injection characteristics of test fuels such as injection rate and mass results are very similar for diesel and biodiesel fuel derived from soybean oil. However, when comparing the start of injection, biodiesel resulted in short injection delay. The Sauter mean diameter (SMD) of biodiesel fuel is higher than that of diesel fuel, and biodiesel showed longer spray tip penetration as compared to that of diesel fuel. Combustion of diesel and biodiesel fuels indicated similar patterns of combustion pressure and rate of heat release. However, the combustion of biodiesel showed lower peak combustion pressures and peak heat release rates than those of diesel fuel becase of its LHV (lower heating value). The comparison of emission characteristics of biodiesel and diesel fuel shows that the biodiesel fuel emitted higher indicated specific NO., and a remarkably low level of soot emissions.