Nipah virus (NiV) is a highly pathogenic emergent paramyxovirus causing deadly encephalitis in humans. Its replication requires a constant supply of unassembled nucleoprotein (N(0)) in complex with its viral chaperone, the phosphoprotein (P). To elucidate the chaperone function of P, we reconstituted NiV the N(0)-P core complex and determined its crystal structure. The binding of the N-terminal region of P blocks the polymerization of N by interfering with subdomain exchange between N protomers and keeps N(0) in an open conformation, ready to grasp an RNA molecule. We found that a peptide derived from the N-binding region of P protects cells against viral infection and demonstrated by structure-based mutagenesis that this peptide acts by inhibiting N(0)-P formation. These results provide new insights about the assembly of N along genomic RNA and validate the N(0)-P complex as a target for drug development.