Biodiesel is an environmentally friendly alternative diesel fuel consisting of the alkyl monoesters of fatty acids. It is obtained from triglycerides through the transesterification process. Biodiesel has been observed to reduce most engine exhaust pollutant emissions with the exception of oxides of nitrogen (NOx), which generally increase by 5% to 15%. The objective of this research was to determine the relationship between changes in combustion timing caused by differences in the fuel properties between diesel fuel and biodiesel and the NOx increase. The properties investigated in this research included the lower heating value, density, speed of sound, bulk modulus, and cetane number of biodiesel. It was found that half of the start of combustion advance associated with biodiesel originated from a start of injection advance that was split approximately evenly between the automatic timing advance of the pump as it injects more fuel to compensate for the lower heating value of biodiesel and the effect of the bulk modulus, viscosity, and density of the fuel. At the same temperature, the fuel delivery of biodiesel was higher than for petroleum-based diesel fuel because of the higher viscosity of biodiesel. At the same viscosity level, it was found that the fuel delivery of petroleum-based diesel fuel was higher than for biodiesel. This was attributed to the metering orifices in the fuel injection pumps restricting the amount of fuel flow for denser fuels. The other half of the start of combustion timing advance was due to the higher cetane number of the biodiesel.