Significance Amyloid deposition of α-syn is a hallmark of PD. Heterozygous point mutations of α-syn are associated with the early-onset and rapid progression of fPD. In this study, we found the fibril strain formed by a familial mutant hE46K induced early-onset motor deficit and enhanced α-syn aggregation in vivo. More importantly, we showed through cryogenic electron microscopy and in vivo studies the E46K fibril strain can template wild-type α-syn monomer to form amyloid fibrils, which inherit both the structure and propagation potency of the template. Our work reveals the structural basis underlying the cross-seeding between wild-type and mutant α-syn, underscores the importance of fibril structure in determining α-syn neuropathology, and provides mechanistic understanding of the pathology of E46K-associated fPD.