Linear double-stranded DNA (dsDNA) viruses package their genome into a procapsid using an ATP-driven nanomotor. Here we report that bacteriophage phi29 DNA packaging motor exercises a one-way traffic property for dsDNA translocation from N-terminal entrance to C-terminal exit with a valve mechanism in DNA packaging, as demonstrated by voltage ramping, electrode polarity switching, and sedimentation force assessment. Without the use of gating control as found in other biological channels, the observed single direction dsDNA transportation provides a novel system with a natural valve to control dsDNA loading and gene delivery in bioreactors, liposomes, or high throughput DNA sequencing apparatus.