The share of biofuels in the transportation sector is increasing. Previous studies revealed that the use of biofuels decreases the size of particles (which is linked to an increase in particulate toxicity). Current emission regulations do not consider small particles (sub-23 nm); however, there is a focus in future emissions regulations on small particles. These and the fact that within cold-start emissions are higher than during the warmed-up operation highlight the importance of a research that studies particulate matter emissions during cold-start. This research investigates the influence of biofuel on PN and PM concentration, size distribution, median diameter and cumulative share at different size ranges (including sub-23 nm and nucleation mode) during cold-start and warm-up operations using diesel and 10, 15 and 20% mixture (coconut biofuel blended with diesel). During cold-start, between 19 and 29% of total PN and less than 0.8% of total PM were related to the nucleation mode (sub-50 nm). Out of that, the share of sub-23 nm was up to 9% for PN while less than 0.02% for PM. By using biofuel, PN increased between 27 and 57% at cold-start; while, the increase was between 4 and 19% during hot-operation. The median diameter also decreased at cold-start and the nucleation mode particles (including sub-23 nm particles) significantly increased. This is an important observation because using biofuel can have a more adverse impact within cold-start period which is inevitable in most vehicles’ daily driving schedules.