This investigation employed the diffused back-illumination (DBI) technique to analyze the spray characteristics of hydrotreated vegetable oil (HVO) fuel at three injection pressures and compared them with conventional diesel fuel. The results showed that as the injection pressure increased, the peak injection rates of both the HVO and diesel increased. At injection pressures above 120 MPa, the injection rates of both fuels were nearly identical, though differences were observed at lower pressures. Increasing the injection pressure reduced the injection delay. The HVO fuel exhibited a shorter spray tip penetration, lower equivalence ratio, larger spray angle, and spray volume, but its spray angle stability was lower than that of diesel. The ambient gas entrainment rate primarily occurred in two stages, significantly influenced by the spray breakup development stage. For diesel sprays, the injection pressure mainly affected the equivalence ratio near the nozzle with minimal downstream impact. Dent’s model provided better predictions of the penetration distance for diesel, while Hiroyasu’s model was more accurate in predicting the penetration distance of the HVO at 120 MPa and 180 MPa. Inagaki’s model performed better in predicting the spray angle for diesel, whereas Hiroyasu’s model was more accurate for the HVO spray angle predictions. Through this research, a better understanding of the spray characteristics of green fuels will be achieved, providing a reference for the design and optimization of new generation engines.