Biomotors are involved in countless vital active processes including muscle motion, heart beating, lung breathing, DNA replication, cell division and viral DNA packaging. In this report, the sequential action of the ATPase ring in the dsDNA packaging motor of phi29 is revealed to be regulated by an arginine finger. Arginine finger is shown to extend from one ATPase subunit to the adjacent one for a noncovalent dimer formation, and is involved in ATP binding, hydrolysis, and DNA translocation. Dimer formation is observed when arginine mutants were mixed with wild-types, which can offer their arginine to promote the inter-subunit interaction. Ultracentrifugation and in vitro virion assembly assays indicated that the ATPase was presenting as monomers and dimer mixtures, based on the results that the isolated dimers alone were deficient in DNA translocation, but the addition of monomer could restore the activity. Moreover, ATP binding or hydrolysis induce two rounds of conformational entropy changes of the ATPase with high or low DNA affinity. Taken together, we concluded that the arginine finger regulates sequential action of the motor ATPase subunit by promoting the formation of the dimer inside the hexamer. The formation of one dimer and four monomers inside the hexamer lead to an asymmetrical configuration of the hexameric ATPase complex. Such organization is supported by structural evidences of many other ATPase systems. All the results above provide clues for why the hexameric phi29 ATPase was previously reported as a pentameric configuration by cryo-electron microscopy (cryo-EM). Since the bridging by the arginine finger renders two adjacent ATPase subunits closer than other subunits, thus, the asymmetrical hexamer would appear as a pseudo-pentamer by cryo-EM, a technology that acquires the average of numerous images.