It was observed that the cement line (CL), namely the border of the osteon in cortical bone, plays an important role in bone fracture: arresting and deflecting cracks. The underlying mechanism was speculated to be that each CL behaves as a weak interface, and thus, it attracts and deflects the bone cracks due to the debonding at the CL. This speculation of a weak CL has not been experimentally verified due to the CL’s challengingly small width. In this study, nanoindentation arrays were carefully conducted to characterize the CLs in ovine and bovine femurs. We found that the modulus and hardness of the CLs are about 30% less than those of the surrounding bone tissues in both species. Thus, for the first time, we characterized the mechanical properties of the CL and verified the speculation of a weak CL, providing a quantitative/constitutive basis for the theoretical modeling of bone micromechanics involving the CL.