Incoherent twin boundaries (ITBs) are widespread and play a crucial role in unidirectional deformation behavior of materials, however, the intrinsic role of individual ITB under cyclic loading remains elusive. Here we show the fatigue cracking behavior of Cu bicrystal with an ITB as its sole interface for the first time. The slip bands (SBs) could transfer through the ITB; meanwhile, the ITB could migrate with the motion of partial dislocations. Both the penetrability and mobility contribute to the higher fatigue cracking resistance of the ITB and hence the fatigue crack nucleates along the SBs preferentially. These new findings not only shed light on the fatigue cracking mechanisms of a penetrable boundary with direct evidence but also could provide important implications for future interfacial optimization of metallic materials.