This study deals with the investigation about the effect of the pilot and split injection strategies on the spray-atomization characteristics of biodiesel fuel derived from a soybean oil. Experimental results were compared with the calculation results obtained from the numerical analysis. Fuel properties of biodiesel according to the variation of the fuel temperature were inserted to the fuel library in the KIVA code. The amount of fuel injection is divided into equal mass for each split and main injection. In this work, the pilot injection strategy can be achieved by the amount of fuel injection shortly before the start of the main injection. A spray tip penetration, radial distance and spray area were measured for the analysis of macroscopic spray characteristics. In addition, the local and overall droplet size distribution were calculated by using KIVA-3V code to study the effect of split and pilot injection on the atomization performance under high ambient pressure. From these studies, the experimental results showed the multiple injection induced the decrease of the spray tip penetration due to the reduction and division of the spray momentum compared to single injection. In the atomization performance, the droplet size increased in the case of the multiple injection a little. Moreover, the SMD slightly increased as the fuel droplets goes through the axial direction. The spray behavior of numerical results were well predicted the experimental multiple spray characteristics of biodiesel fuel.