Nanoindentation measurements were performed for Fe-C based martensitic steels, and then the strengthening factors such as grain boundary effect were evaluated. Nanohardness of the matrix of the martensite is lower than that expected from macroscopic hardness, indicating that the grain boundary effect is significant for the macroscopic strength of the Fe-C martensite. A remarkable decrease of the grain boundary effect was found at the tempering temperature of 673 K, which is due to a disappearance of film-like carbides on grain boundaries. These results will be discussed in light of the interpretations of grain boundary strengthening.