We have synthesized a new amphiphilic DNA containing an internal hydrophobic region consisting of dodecyl phosphotriester linkages (dod-DNA). dod-DNAs of any arbitrary sequence can be synthesized by standard automated solid-phase synthesis using a set of nucleoside dodecyl phosphoramidites. Although replacement of a phosphodiester linkage with the hydrophobic phosphotriester linkage tended to decrease the thermodynamic stability of the duplex, when multiple dodecyl groups were introduced into both strands of a DNA duplex in a face-to-face arrangement, the resulting duplex was strongly stabilized by the interstrand hydrophobic interaction. The hydrophobic zipper formation provides a tightly associated, electroneutral, and highly hydrophobic region in the originally hydrophilic DNA duplex. As the hydrophobic properties can be adjusted by varying the encoded sequence, dod-DNA is an easy-to-design amphiphilic molecule for controllable vesicles and membrane biotechnology.