Double-stranded (ds)DNA viruses package their genomic DNA into a procapsid using a force-generating nanomotor powered by ATP hydrolysis. Viral DNA packaging motors are mainly composed of the connector channel and two DNA packaging enzymes. In 1998, it was proposed that viral DNA packaging motors exercise a mechanism similar to the action of AAA+ ATPases that assemble into ring-shaped oligomers, often hexamers, with a central channel (Guo et al. Molecular Cell, 2:149). This chapter focuses on the most recent findings in the bacteriophage ϕ29 DNA packaging nanomotor to address this intriguing notion. Almost all dsDNA viruses are composed entirely of protein, but in the unique case of ϕ29, packaging RNA (pRNA) plays an intermediate role in the packaging process. Evidence revealed that DNA packaging is accomplished via a "push through one-way valve" mechanism. The ATPase gp16 pushes dsDNA through the connector channel section by section into the procapsid. The dodecameric connector channel functions as a one-way valve that only allows dsDNA to enter but not exit the procapsid during DNA packaging. Although the roles of the ATPase gp16 and the motor connector channel are separate and independent, pRNA bridges these two components to ensure the coordination of an integrated motor. ATP induces a conformational change in gp16, leading to its stronger binding to dsDNA. Furthermore, ATP hydrolysis led to the departure of dsDNA from the ATPase/dsDNA complex, an action used to push dsDNA through the connector channel. It was found unexpectedly that by mutating the basic lysine rings of the connector channel or by changing the pH did not measurably impair DNA translocation or affect the one-way traffic property of the channel, suggesting that the positive charges in the lysine ring are not essential in gearing the dsDNA. The motor channel exercises three discrete, reversible, and controllable steps of gating, with each step altering the channel size by 31% to control the direction of translocation of dsDNA. Many DNA packaging models have been contingent upon the number of base pairs packaged per ATP relative to helical turns for B-type DNA. Both 2 and 2.5 bp per ATP have been used to argue for four, five, or six discrete steps of DNA translocation. The "push through one-way valve" mechanism renews the perception of dsDNA packaging energy calculations and provides insight into the discrepancy between 2 and 2.5 bp per ATP. Application of the DNA packaging motor in nanotechnology and nanomedicine is also addressed. Comparison with nine other DNA packaging models revealed that the "push through one-way valve" is the most agreeable mechanism to interpret most of the findings that led to historical models. The application of viral DNA packaging motors is also discussed.