The present study investigated the effects of adding 20 vol.% biodiesel to petroleum diesel (to produce a mixture termed B20) on the physical properties and reactivity of the resulting exhaust soot particles. Tests were performed at different engine loads of a constant speed, and the soot particles from the combustion of B20 and petroleum diesel fuel (DF) were collected from the engine exhaust stream. Transmission electron microscopy and Raman spectroscopy were employed for the analysis of soot morphology and nanostructure. The thermogravimetric analysis was used to determine the oxidative reactivity of the soot. For both the DF and B20 soot, increased engine loads result in soot aggregates with more compact morphology and primary soot particles with larger size and more organized structure. Compared to the DF soot, the B20 aggregates have a slightly more compact morphology and smaller primary particle size. No appreciable differences are observed in nanostructure between the DF and B20 soot. The thermogravimetric analysis demonstrates that the B20 soot is associated with lower peak temperature, burnout temperature and apparent activation energy, suggesting that it is more reactive than the DF soot.