While the prion protein (PrP) is clearly involved in neuropathology, its physiological roles remain elusive. Here, we demonstrate PrP functions in cell–substrate interaction in Drosophila S2, N2a and HeLa cells. PrP promotes cell spreading and/or filopodia formation when overexpressed, and lamellipodia when downregulated. Moreover, PrP normally accumulates in focal adhesions (FAs), and its downregulation leads to reduced FA numbers, increased FA length, along with Src and focal adhesion kinase (FAK) activation. Furthermore, its overexpression elicits the formation of novel FA‐like structures, which require intact reggie/flotillin microdomains. Altogether, PrP modulates process formation and FA dynamics, possibly via signal transduction involving FAK and Src.