Z-DNA is a non-canonical, left-handed helical structure that plays crucial roles in various cellular processes. DNA mismatches, which involve the incorporation of incorrect Watson-Crick base pairs, are present in all living organisms and should contribute to the mechanism of Z-DNA formation. However, the impact of mismatches on Z-DNA formation remains poorly understood. Moreover, the combined effect of DNA mismatches and bending, a common biological phenomenon observed in vivo, has not yet been explored due to technological limitations. Here, using single-molecule FRET, we show that a mismatch inside Z-DNA region, i.e., the CG repeat region, hinders Z-DNA formation. In stark contrast, however, a mismatch in the B-Z junction facilitates Z-DNA formation. When the bending force is applied on double stranded DNA, a mismatch in the B-Z junction releases the bending stress more effectively than one in the CG repeat region. These findings provide mechanical insights into the role of DNA mismatches and bending forces in regulating Z-DNA formation, whether promoting or inhibiting it in biological environments.