An intron is an extended genomic feature whose function requires multiple constrained positions--donor and acceptor splice sites, a branch point, a polypyrimidine tract and suitable splicing enhancers--that may be distributed over hundreds or thousands of nucleotides. New introns are therefore unlikely to emerge by incremental accumulation of functional sub-elements. Here we demonstrate that a functional intron can be created de novo in a single step by a segmental genomic duplication. This experiment recapitulates in vivo the birth of an intron that arose in the ancestral jawed vertebrate lineage nearly half-a-billion years ago.