Finite fossil energy sources and carbon dioxide as a main cause for climate changes are still under critical discussion. Therefore, scientists work on the replacement of fossil by alternative diesel fuels from biomass. Hence, in this study the in-cylinder combustion and particle number emissions of di-n-butyl ether (DNBE), as a representative of second generation biofuels, and of reference diesel fuel (B0) for comparison were analyzed by several measurement techniques at different injection and boost pressures. The heat release rate and thus the ignition delay as well as the center of combustion were analyzed by monitoring the global in-cylinder pressure signal using a pressure sensor. The combustion process was also visualized by simultaneous imaging of the hydroxyl radical and a spectral range of soot luminescence. This allows the analysis of the in-cylinder soot formation and oxidation process. Changes in physical properties of the emitted particles were measured by a Scanning Mobility Particle Sizer. Our measurements show a much more homogeneous combustion for DNBE compared to B0. This can be explained by the dissimilar fuel properties of DNBE in comparison to B0 causing significant differences in in-cylinder mixture formation. As a consequence of the nearly homogeneous combustion of DNBE, particle number emissions are considerably lower and the size of the emitted particles is smaller than for B0.