It has been recognized that density, viscosity, surface tension, and volatility of liquid fuel are of great importance on the atomization and vaporization characteristics of biodiesel spray. This paper presents a comprehensive physical property prediction of biodiesel fuel for spray modeling with most recently developed property prediction models. The temperature-dependent properties of a soy methyl ester (SME) biodiesel were well predicted by the updated prediction methods. Then, the physical properties of the SME biodiesel were added into the KIVA-3V fuel library. By using the well predicted fuel properties, the spray behaviors of SME were successfully simulated by the KIVA-3V code under late-cycle post-injection, conventional diesel injection, and early-injection engine-relevant conditions. The simulation results agree reasonably well with the available experimental liquid penetrations under conditions of various ambient densities and temperatures. The different properties between diesel and SME fuels have pronounced effect on their different spray behaviors under the late-cycle post-injection conditions. Under the conventional diesel injection conditions, the much higher ambient density with moderately high temperature reduces the effect of the different properties between the two fuels on their different spray characteristics. In contrast, the lower temperature enhances the effect of the different properties between the two fuels on their different spray behaviors under the early-injection conditions.