Surface topography and compression elasticity of bovine cardiac muscle fibers in rigor and relaxing state has been studied with atomic force microscopy. Characteristic sarcomere patterns running along the longitudinal axis of the fibers were clearly observed, and Z-lines, M-lines, I-bands, and A-bands can be distinguished through comparing with TEM images and force curves. AFM height images of fibers had shown a sarcomere length of 1.22±0.02μm (n=5) in rigor with a significant 9% increase in sarcomere length in relaxing state (1.33±0.03μm, n=5), indicating that overlap move with the changing physiological conditions. Compression elasticity curves along with sarcomere locations have been taken by AFM compression processing. Coefficient of Z-line, I-band, Overlap, and M-line are 25±2pN/nm, 8±1pN/nm, 10±1pN/nm, and 17±1.5pN/nm respectively in rigor state, and 18±2.5pN/nm, 4±0.5pN/nm, 6±1pN/nm, and 11±0.5pN/nm respectively in relaxing state. Young’s Modulus in Z-line, I-band, Overlap, and M-line are 115±12kPa, 48±9kPa, 52±8kPa, and 90±12kPa respectively in rigor, and 98±10kPa, 23±4kPa, 42±4kPa, and 65±7kPa respectively in relaxing state. The elasticity curves has shown a similar appearance to the section analysis profile of AFM height images of sarcomere and the distance between adjacent largest coefficient and Young’s Modulus is equal to the sarcomere length measured from the AFM height images using section analysis, indicating that mechanic properties of fibers have a similar periodicity to the topography of fibers.