The cellular prion protein (PrPC), encoded by the PRNP gene, is a ubiquitous glycoprotein, which is highly expressed in the brain. This protein, mainly known for its role in neurodegenerative diseases, is involved in several physiological processes including neurite outgrowth. By using a novel focal stimulation technique, we explored the potential function of PrPC, in its soluble form, as a signaling molecule. Thus, soluble recombinant prion proteins (recPrP) encapsulated in micro-vesicles were released by photolysis near the hippocampal growth cones. Local stimulation of wild-type growth cones with full-length recPrP induced neurite outgrowth and rapid growth cone turning towards the source. This effect was shown to be concentration dependent. Notably, PrPC-knockout growth cones were insensitive to recPrP stimulation, but this property was rescued in PrPknockout growth cones expressing GFP-PrP. Taken together, our findings indicate that recPrP functions as a signaling molecule, and that its homophilic interaction with membrane-anchored PrPC might promote neurite outgrowth and facilitate growth cone guidance. © 2016. Published by The Company of Biologists Ltd.