Gallium nitride (GaN)-based nanowires (NWs) have attracted much attention for the fabrication of novel nanostructured devices. In this paper, the influence of an AlN seeding layer on the nucleation of self-assembled GaN NWs grown by plasma-assisted molecular beam epitaxy (MBE) on Si (111) substrates has been investigated. Not only is the formation of a two-dimensional compact GaN layer at the bottom of the NWs suppressed, but also a high density of vertically aligned well-separated GaN NWs originating from GaN islands are successfully obtained after introducing annealing and nitridation processes. Scanning electronic microscope and transmission electron microscope measurements show that the NWs have a high crystalline wurtzite structure nearly free of dislocations and stacking faults and the NW diameter remains constant over almost the entire length. Due to the temperature-dependent diffusion length of Ga adatoms during the nucleation process, the formation of well-separated NWs relies on the distribution and morphology of the underlying AlN seeding layer. Moreover, the SiNx layer served as mask to inhibit coalescence at the nucleation sites. The developed growth processes and the obtained results provide a viable path facilitating the use of MBE growth techniques to fabricate III-nitride NW-based materials and related devices on Si substrates.