There are significant differences in mechanical stability and unfolding dynamics among proteins with different structural compositions. Compared with proteins with β-sheets and subjected to shearing forces, proteins that are composed entirely of α-helices often undergo rapid unfolding under low stretching forces, thus requiring quantitative studies by using experimental tools that can precisely control forces on a pico-Newton scale. Magnetic tweezers with intrinsic force-control capability and great stability for long-time continuous measurement are suitable to measure force-induced conformation transitions of protein subjected to low forces of several pico-Newton. Acyl-CoA binding protein (ACBP) is a model protein used to study the folding/unfolding kinetics of complete α-helices protein. It is composed of 86 amino acid residues, forming a helical bundle of four α-helices. When its N- and C-terminal are stretched, the first and last α-helix are subjected to shear force in parallel. Previous biochemical studies showed that ACBP folding and unfolding in a two-state manner. In this paper, we use magnetic tweezers to stretch ACBP from its N- and C-end and obtain the distribution of the unfolding force at different loading rates ranging from 0.25 pN/s to 4 pN/s. The most probable unfolding forces are all less than 10 pN, which confirms that ACBP is not mechanically stable. At a constant loading rate, the unfolding force distribution and the most probable unfolding force as a function of loading rate have well-defined analytical formulas based on Bell’s model. Therefore, the experimental results of unfolding force can be fitted directly to obtain the important kinetic parameter of unfolding distance which is defined as the difference in extension between the native state and the transition state. Data analysis shows that ACBP has an extraordinarily long unfolding distance of 7.8 nm. Steered molecular dynamics simulations of ACBP stretching gives the transition state with N-terminal α-helix fully unfolded and C-terminal α-helix partially unfolded, which is consistent with the long unfolding distance obtained in the experiment on magnetic tweezers. According to the simulation results, the unfolding of α-helices is less cooperative than that of β-sheet structures. This characteristic makes α-helix proteins sensitive to mechanical forces, rendering them suitable as force sensors in cells. This study shows that single-molecule stretching experiment combined with molecular dynamics simulations is a reliable method to reveal the molecular mechanism of protein conformationtransitions under stretching forces.