α-Tropomyosin (αTm) is the predominant tropomyosin isoform in adult human heart and constitutes a major component in Ca2+-regulated systolic contraction of cardiac muscle. The familial hypertrophic cardiomyopathy (FHC)-related αTm E180G mutant is associated with decreased thermal stability and enhanced Ca2+-sensitivity in functional assays. A mechanistic relationship between the E180G missense mutation and functional changes has not been established. We present here the first direct probe images of WT human cardiac and E180G mutant αTm by atomic force microscopy, and demonstrate that the mutant is more flexible than WT. Single molecules of bacterially-expressed human cardiac αTm were imaged on poly-lysine coated mica and their contours were analyzed. Analysis of tangent correlation along molecular contours yielded values of persistence length (Lp), which showed 27% increase in flexibility of the E180G mutant. Increased flexibility of the mutant was confirmed by shorter mean end-to-end length and fitting end-to-end length distributions to the worm-like chain model. Corresponding to increased flexibility, it is expected that less mechanical moment, and hence a lesser extent of Ca2+-induced conformational change of troponin, are required to perturb αTm to initiate thin filament activation during systole, thus explaining enhanced Ca2+-sensitivity of function. Hypersensitivity to Ca2+ could overwork cardiac muscle resulting in hypertrophy in FHC. Support: NIH and American Heart Association.