The mechanical conditions of fatigue crack initiation were evaluated on roughly 500 nm thick freestanding copper films. Fatigue experiments and fatigue damage observations by using field emission scanning electron microscope (FESEM) and electron backscatter diffraction (EBSD) analysis revealed that an intrusion/extrusion formed parallel to a Σ3 twin boundary, and fatigue crack then initiated from this intrusion/extrusion. On the basis of these results, anisotropic elastic finite element method (FEM) analysis considering grain shape and crystal orientation around the fatigue crack initiation sites was conducted in order to evaluate local stress distribution near fatigue crack initiation sites. FEM analysis results indicated that fatigue crack initiation occurred at the slip system which penetrated the film in thickness direction. Moreover, number of cycles to fatigue crack initiation showed a tendency to increase as resolved shear stress decreased.