Z-DNA binding proteins (ZBPs) specifically recognize and stabilize left-handed double helices, including Z-DNA and Z-RNA. However, the energetics of Z-form stabilization by ZBPs have never been characterized due to the technical limitations of bulk studies, resulting in an unclear understanding of the ZBP operational mechanism at the molecular level. Here, we use single-molecule fluorescence resonance energy transfer (FRET) to determine the energetics of Z-form stabilization by ZBP for DNA, RNA, and DNA-RNA duplexes, revealing that the formation of B-Z or A-Z junctions dominates the thermodynamics and kinetics of Z-form stabilization. Furthermore, in contrast to general assumptions, the Z-form is most efficiently and most rapidly formed in the DNA-RNA hybrid duplex due to the greatly reduced junction energy in the DNA-RNA hybrid.