Diesel engines are one of the most important power generating units these days. Increasing emission levels, climate change and the need for energy security has prompted increasing research into alternative fuels for diesel engines. Biodiesel is the most popular amongst the alternatives for diesel fuel as it is biodegradable, renewable and can be produced domestically from vegetable oils. In recent years, hydro-treated vegetable oil (HVO) has also gained popularity due to some of its advantages over biodiesel such as higher cetane number, lower deposit formation, storage stability etc. HVO is a renewable, paraffinic biobased alternative fuel for diesel engines similar to biodiesel. Unlike biodiesel, production process for HVO involves hydrogen as catalyst instead of methanol which removes oxygen content from vegetable oil. A modified 6-cylinder heavy-duty diesel engine (modified for operation with single cylinder) was used for studying particle number emission characteristics for HVO fuel. The investigation was performed for varying fuel injection pressure at various engine operating loads (6, 8, 10, 12 and 14 bar IMEP). Five rail pressures were chosen from 800 to 2000 bar at a step of 300 bar. The results show that increasing rail pressure tend to increase nucleation mode particle concentration while engine load resulted in much higher total particle number concentration. No significant differences were observed in soot and oxides of nitrogen (NOx) emission for HVO compared to mineral diesel. It was also observed that nucleation mode particles are prone to contribute more compared to accumulation mode particles with increasing engine IMEP. In this study, a comparative investigation has been performed for engine out emissions for diesel, rapeseed methyl ester (RME) and HVO. A 6-cylinder heavy duty Scania engine modified for single cylinder operation has been used for the study. Experiments have been performed with varying injection timings and EGR ratio at 5 bar imep, 1200 rpm engine speed. The main focus is to evaluate NOx-particulate matter trade-off along with the measurement of other regulated emissions for the three fuels. A correlation between particulate mass and particulate number emission has also been investigated. The study shows the potential for the utilization of HVO in comparison to diesel and RME biodiesel.